Diagnostic antibody assay

ABSTRACT

The present invention pertains to novel diagnostic assays for the diagnosis of amyloidosis, in particular Alzheimer&#39;s disease, and related aspects. In particular, monoclonal antibodies and an antibody assay are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/082,309, filed on Jul. 21, 2008, which is incorporated herein byreference in its entirety for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN COMPUTER READABLEFORMAT (CRF)

The Sequence Listing, which is a part of the present disclosure,includes a computer readable form and a written sequence listingcomprising nucleotide and/or amino acid sequences of the presentinvention. The sequence listing information recorded in computerreadable form is identical to the written sequence listing. The subjectmatter of the Sequence Listing is incorporated herein by reference inits entirety.

INCORPORATION-BY-REFERENCE OF DEPOSITED BIOLOGICAL MATERIAL

The following biological material, which is a part of the presentdisclosure, has been deposited in accordance with the Budapest Treatyand are available at the Deutsche Sammlung für Mikroorganismen undZellkulturen (DSMZ), Inhoffenstr. 7b, 38124 Braunschweig, DE: hybridomacell line 5-5-6, Deposit No. DSM ACC 2923, deposit date Jun. 17, 2008;hybridoma cell line 6-1-6, Deposit No. DSM ACC 2924, deposit date Jun.17, 2008; hybridoma cell line 17-4-3, Deposit No. DSM ACC 2925, depositdate Jun. 17, 2008; and hybridoma cell line 24-2-3, Deposit No. DSM ACC2926, deposit date Jun. 17, 2008. Such hybridoma cell lines can producemonoclonal antibodies specifically recognizing Aβ N3pE-x. The depositedbiological material described above is incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

The present invention pertains to novel diagnostic assays for thediagnosis of amyloidosis, a group of disorders and abnormalitiesassociated with amyloid protein such as Alzheimer's disease and relatedaspects. In particular, an antibody assay is provided.

BACKGROUND OF THE INVENTION

Amyloidosis is not a single disease entity but rather a diverse group ofprogressive disease processes characterized by extracellular tissuedeposits of a waxy, starch-like protein called amyloid, whichaccumulates in one or more organs or body systems. As the amyloiddeposits accumulate, they begin to interfere with the normal function ofthe organ or body system. There are at least 15 different types ofamyloidosis. The major forms are primary amyloidosis without knownantecedent, secondary amyloidosis following some other condition, andhereditary amyloidosis.

Secondary amyloidosis occurs during chronic infection or inflammatorydisease, such as tuberculosis, a bacterial infection called familialMediterranean fever, bone infections (osteomyelitis), rheumatoidarthritis, inflammation of the small intestine (granulomatous ileitis),Hodgkin's disease and leprosy.

Amyloid deposits include amyloid P (pentagonal) component (AP), aglycoprotein related to normal serum amyloid P (SAP), and sulphatedglycosaminoglycans (GAG), complex carbohydrates of connective tissue.Amyloid protein fibrils, which account for about 90% of the amyloidmaterial, comprise one of several different types of proteins. Theseproteins are capable of folding into so-called “beta-pleated” sheetfibrils, a unique protein configuration which exhibits binding sites forCongo red resulting in the unique staining properties of the amyloidprotein.

Many diseases of aging are based on or associated with amyloid-likeproteins and are characterized, in part, by the buildup of extracellulardeposits of amyloid or amyloid-like material that contribute to thepathogenesis, as well as the progression of the disease. These diseasesinclude, but are not limited to, neurological disorders such as mildcognitive impairment (MCI), Alzheimer's disease (AD), like for instancesporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias(FAD) like Familial British Dementia (FBD) and Familial Danish Dementia(FDD), neurodegeneration in Down Syndrome, Lewy body dementia,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex. Other diseases which are based on orassociated with amyloid-like proteins are progressive supranuclearpalsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson'sdisease, HIV-related dementia, ALS (amyotropic lateral sclerosis), AdultOnset Diabetes; senile cardiac amyloidosis; endocrine tumors, andothers, including macular degeneration.

Although pathogenesis of these diseases may be diverse, theircharacteristic deposits often contain many shared molecularconstituents. To a significant degree, this may be attributable to thelocal activation of pro-inflammatory pathways thereby leading to theconcurrent deposition of activated complement components, acute phasereactants, immune modulators, and other inflammatory mediators (McGeeret al., Tohoku J Exp Med. 174(3): 269-277 (1994)).

Recently, accumulating evidence demonstrates involvement of N-terminalmodified Aβ peptide variants in Alzheimer's disease. Aiming biopsiesdisplay a presence of Aβ 1-40 and Aβ 1-42 not only in the brain ofAlzheimer's patients but also in senile plaques of unaffectedindividuals. However, N-terminal truncated and pyroGlu modified AβN3pE-40/Aβ N3pE-42 is almost exclusively engrained within plaques ofAlzheimer's disease patients, making this Aβ variant an eligiblediagnostic marker and a potential target for drug development.

At present, several commercial manufacturers offer ELISA kits whichallow a detection of Aβ 1-40/1-42 and Aβ N3pE-40/Aβ N3pE-42 in the lowpicogramm (pg) range.

The brains of Alzheimer's disease (AD) patients are morphologicallycharacterized by the presence of neurofibrillary tangles and by depositsof Aβ peptides in neocortical brain structures (Selkoe, D. J. & Schenk,D. Alzheimer's disease: molecular understanding predicts amyloid-basedtherapeutics. Annu. Rev. Pharmacol. Toxicol. 43, 545-584 (2003)). Aβpeptides are liberated from the amyloid precursor protein (APP) aftersequential cleavage by β- and γ-secretase. The γ-secretase cleavageresults in the generation of Aβ 1-40 and Aβ 1-42 peptides, which differin their C-termini and exhibit different potencies of aggregation,fibril formation and neurotoxicity (Shin, R. W. et al. Amyloidbeta-protein (Abeta) 1-40 but not Abeta 1-42 contributes to theexperimental formation of Alzheimer disease amyloid fibrils in ratbrain. J. Neurosci. 17, 8187-8193 (1997); Iwatsubo, T. et al.Visualization of Abeta 42(43) and Abeta 40 in senile plaques withend-specific Abeta monoclonals: evidence that an initially depositedspecies is Abeta 42(43). Neuron 13, 45-53 (1994); Iwatsubo, T., Mann, D.M., Odaka, A., Suzuki, N. & Ihara, Y. Amyloid beta protein (Abeta)deposition: Abeta 42(43) precedes Abeta 40 in Down syndrome. Ann.Neurol. 37, 294-299 (1995); Hardy, J. A. & Higgins, G. A. Alzheimer'sdisease: the amyloid cascade hypothesis. Science 256, 184-185 (1992);Roβner, S., Ueberham, U., Schliebs, R., Perez-Polo, J. R. & Bigl, V. Theregulation of amyloid precursor protein metabolism by cholinergicmechanisms and neurotrophin receptor signaling. Prog. Neurobiol. 56,541-569 (1998)). In addition to C-terminal variability, N-terminallymodified Aβ peptides are abundant (Saido, T. C. et al. Dominant anddifferential deposition of distinct beta-amyloid peptide species, A betaN3(pE), in senile plaques. Neuron 14, 457-466 (1995); Russo, C. et al.Presenilin-1 mutations in Alzheimer's disease. Nature 405, 531-532(2000); Saido, T. C., Yamao, H., Iwatsubo, T. & Kawashima, S. Amino- andcarboxyl-terminal heterogeneity of beta-amyloid peptides deposited inhuman brain. Neurosci. Lett. 215, 173-176 (1996)). It appears that amajor proportion of the Aβ peptides undergoes N-terminal truncation bytwo amino acids, exposing a glutamate residue, which is subsequentlycyclized into pyroglutamate (pE), resulting in Aβ3(pE)-42 peptides(Saido, T. C. et al. Dominant and differential deposition of distinctbeta-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron14, 457-466 (1995); Saido, T. C., Yamao, H., Iwatsubo, T. & Kawashima,S. Amino- and carboxyl-terminal heterogeneity of beta-amyloid peptidesdeposited in human brain. Neurosci. Lett. 215, 173-176 (1996)).Alternatively, pE may be formed following β′-cleavage by BACE1,resulting in Aβ N11(pE)-42 (Naslund, J. et al. Relative abundance ofAlzheimer A beta amyloid peptide variants in Alzheimer disease andnormal aging. Proc. Natl. Acad. Sci. U.S.A. 91, 8378-8382 (1994); Liu,K. et al. Characterization of Abeta11-40/42 peptide deposition inAlzheimer's disease and young Down's syndrome brains: implication ofN-terminally truncated Abeta species in the pathogenesis of Alzheimer'sdisease. Acta Neuropathol. 112, 163-174 (2006)). In particular AβN3(pE)-42 has been shown to be a major constituent of Aβ deposits insporadic and familial Alzheimer's disease (FAD) (Saido, T. C. et al.Dominant and differential deposition of distinct beta-amyloid peptidespecies, A beta N3(pE), in senile plaques. Neuron 14, 457-466 (1995);Miravalle, L. et al. Amino-terminally truncated Abeta peptide speciesare the main component of cotton wool plaques. Biochemistry 44,10810-10821 (2005)).

The Aβ N3pE-42 peptides coexist with Aβ 1-40/1-42 peptides (Saido, T. C.et al. Dominant and differential deposition of distinct beta-amyloidpeptide species, Abeta N3pE, in senile plaques. Neuron 14, 457-466(1995); Saido, T. C., Yamao, H., Iwatsubo, T. & Kawashima, S. Amino- andcarboxyl-terminal heterogeneity of beta-amyloid peptides deposited inhuman brain. Neurosci. Lett. 215, 173-176 (1996)), and, based on anumber of observations, could play a prominent role in the pathogenesisof AD. For example, a particular neurotoxicity of Aβ N3pE-42 peptideshas been outlined (Russo, C. et al. Pyroglutamate-modified amyloidbeta-peptides—AbetaN3(pE)—strongly affect cultured neuron and astrocytesurvival. J. Neurochem. 82, 1480-1489 (2002) and the pE-modification ofN-truncated Aβ peptides confers resistance to degradation by mostaminopeptidases as well as Aβ-degrading endopeptidases (Russo, C. et al.Pyroglutamate-modified amyloid beta-peptides—AbetaN3(pE)—strongly affectcultured neuron and astrocyte survival. J. Neurochem. 82, 1480-1489(2002); Saido, T. C. Alzheimer's disease as proteolytic disorders:anabolism and catabolism of beta-amyloid. Neurobiol. Aging 19, S69-S75(1998)). The cyclization of glutamic acid into pE leads to a loss ofN-terminal charge resulting in accelerated aggregation of Aβ N3pEcompared to the unmodified Aβ peptides (He, W. & Barrow, C. J. The Abeta3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque havegreater beta-sheet forming and aggregation propensities in vitro thanfull-length A beta. Biochemistry 38, 10871-10877 (1999); Schilling, S.et al. On the seeding and oligomerization of pGlu-amyloid peptides (invitro). Biochemistry 45, 12393-12399 (2006)). Thus, reduction of AβN3pE-42 formation should destabilize the peptides by making them moreaccessible to degradation and would, in turn, prevent the formation ofhigher molecular weight Aβ aggregates and enhance neuronal survival.

However, for a long time it was not known how the pE-modification of Aβpeptides occurs. Recently, it was discovered that glutaminyl cyclase(QC) is capable to catalyze Aβ N3pE-42 formation under mildly acidicconditions and that specific QC inhibitors prevent Aβ N3pE-42 generationin vitro (Schilling, S., Hoffmann, T., Manhart, S., Hoffmann, M. &Demuth, H.-U. Glutaminyl cyclases unfold glutamyl cyclase activity undermild acid conditions. FEBS Lett. 563, 191-196 (2004); Cynis, H. et al.Inhibition of glutaminyl cyclase alters pyroglutamate formation inmammalian cells. Biochim. Biophys. Acta 1764, 1618-1625 (2006)).

Lewy body dementia (LBD) is a neurodegenerative disorder that can occurin persons older than 65 years of age, and typically causes symptoms ofcognitive (thinking) impairment and abnormal behavioral changes.Symptoms can include cognitive impairment, neurological signs, sleepdisorder, and autonomic failure. Cognitive impairment is the presentingfeature of LBD in most cases. Patients have recurrent episodes ofconfusion that progressively worsen. The fluctuation in cognitiveability is often associated with shifting degrees of attention andalertness. Cognitive impairment and fluctuations of thinking may varyover minutes, hours, or days. Lewy bodies are formed from phosphorylatedand nonphosphorylated neurofilament proteins; they contain the synapticprotein alpha-synuclein as well as ubiquitin, which is involved in theelimination of damaged or abnormal proteins. In addition to Lewy Bodies,Lewy neurites, which are inclusion bodies in the cell processes of thenerve cells, may also be present. Amyloid plaques may form in the brainsof patients afflicted with DLB, however they tend to be fewer in numberthan seen in patients with Alzheimer's disease. Neurofibrillary tangles,the other micropathological hallmark of AD, are not a maincharacteristic of LBD but are frequently present in addition to amyloidplaques.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration ofupper and lower motor neurons. In some ALS patients, dementia or aphasiamay be present (ALS-D). The dementia is most commonly a frontotemporaldementia (FTD), and many of these cases have ubiquitin-positive,tau-negative inclusions in neurons of the dentate gyrus and superficiallayers of the frontal and temporal lobes.

Inclusion-body myositis (IBM) is a crippling disease usually found inpeople over age 50, in which muscle fibers develop inflammation andbegin to atrophy—but in which the brain is spared and patients retaintheir full intellect. Two enzymes involved in the production ofamyloid-β protein were found to be increased inside the muscle cells ofpatients with this most common, progressive muscle disease of olderpeople, in which amyloid-β is also increased.

Another disease that is based on or associated with the accumulation anddeposit of amyloid-like protein is macular degeneration. Maculardegeneration is a common eye disease that causes deterioration of themacula, which is the central area of the retina (the paper-thin tissueat the back of the eye where light-sensitive cells send visual signalsto the brain). Sharp, clear, “straight ahead” vision is processed by themacula. Damage to the macula results in the development of blind spotsand blurred or distorted vision. Age-related macular degeneration (AMD)is a major cause of visual impairment in the United States and forpeople over age 65 it is the leading cause of legal blindness amongCaucasians. Approximately 1.8 million Americans of age 40 and older haveadvanced AMD, and another 7.3 million people with intermediate AMD areat substantial risk for vision loss. The government estimates that by2020 there will be 2.9 million people with advanced AMD. Victims of AMDare often surprised and frustrated to find out how little is known aboutthe causes and treatment of this blinding condition.

There are two forms of macular degeneration: dry macular degenerationand wet macular degeneration. The dry form, in which the cells of themacula slowly begin to break down, is diagnosed in 85 percent of maculardegeneration cases. Both eyes are usually affected by dry AMD, althoughone eye can lose vision while the other eye remains unaffected. Drusen,which are yellow deposits under the retina, are common early signs ofdry AMD. The risk of developing advanced dry AMD or wet AMD increases asthe number or size of the drusen increases. It is possible for dry AMDto advance and cause loss of vision without turning into the wet form ofthe disease; however, it is also possible for early-stage dry AMD tosuddenly change into the wet form.

The wet form, although it only accounts for 15 percent of the cases,results in 90 percent of the blindness, and is considered advanced AMD(there is no early or intermediate stage of wet AMD). Wet AMD is alwayspreceded by the dry form of the disease. As the dry form worsens, somepeople begin to have abnormal blood vessels growing behind the macula.These vessels are very fragile and will leak fluid and blood (hence‘wet’ macular degeneration), causing rapid damage to the macula.

The dry form of AMD will initially often cause slightly blurred vision.The center of vision in particular may then become blurred and thisregion grows larger as the disease progresses. No symptoms may benoticed if only one eye is affected. In wet AMD, straight lines mayappear wavy and central vision loss can occur rapidly.

Diagnosis of macular degeneration typically involves a dilated eye exam,visual acuity test, and a viewing of the back of the eye using aprocedure called fundoscopy to help diagnose AMD, and—if wet AMD issuspected—fluorescein angiography may also be performed. If dry AMDreaches the advanced stages, there is no current treatment to preventvision loss. However, a specific high dose formula of antioxidants andzinc may delay or prevent intermediate AMD from progressing to theadvanced stage. Macugen® (pegaptanib sodium injection), laserphotocoagulation and photodynamic therapy can control the abnormal bloodvessel growth and bleeding in the macula, which is helpful for somepeople who have wet AMD; however, vision that is already lost will notbe restored by these techniques. If vision is already lost, low visionaids exist that can help improve the quality of life.

One of the earliest signs of age-related macular degeneration (AMD) isthe accumulation of extracellular deposits known as drusen between thebasal lamina of the retinal pigmented epithelium (RPE) and Bruch'smembrane (BM). Recent studies conducted by Anderson et al. haveconfirmed that drusen contain amyloid beta. (Experimental Eye Research78 (2004) 243-256).

The aim of the present invention is to establish a highly sensitive andconcomitantly robust detection technique that allows quantitativedetermination of Aβ variants, in particular pGlu-Aβ peptides, inbiological samples, e.g. liquor or serum samples, preferably serumsamples. This is a tremendous challenge, taking the low abundance of Aβpeptides in blood into account. Having such a detection techniqueavailable is, however, a prerequisite for studying efficacy of smallmolecule inhibitors in drug screening programs.

The present invention provides novel methods and compositions comprisinghighly specific and highly effective antibodies, including chimericantibodies and fragments thereof, including partially or fully humanizedantibodies and fragments thereof, having the ability to specificallyrecognize and bind to specific epitopes from a range of β-amyloidantigens, in particular pGlu-Aβ peptides, which may be presented to theantibody in a monomeric, dimeric, trimeric, etc, or a polymeric form, inform of an aggregate, fibers, filaments or in the condensed form of aplaque. The antibodies enabled by the teaching of the present inventionare particularly useful for diagnosis of amyloidosis, a group ofdiseases and disorders associated with amyloid plaque formationincluding secondary amyloidosis and age-related amyloidosis including,but not limited to, neurological disorders such as Alzheimer's Disease(AD), Lewy body dementia, Down's syndrome, hereditary cerebralhemorrhage with amyloidosis (Dutch type); the Guam Parkinson-Dementiacomplex; as well as other diseases which are based on or associated withamyloid-like proteins such as progressive supranuclear palsy, multiplesclerosis; Creutzfeld Jacob disease, hereditary cerebral hemorrhage withamyloidosis Dutch type, Parkinson's disease, HIV-related dementia, ALS(amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiacamyloidosis; endocrine tumors, and others, including maculardegeneration, to name just a few.

To meet all demands mentioned above, an ELISA based technique would beespecially preferable. The task was started with AP N3pE ELISA, becausefor this Aβ variant an ELISA system is already commercially available(Human Amyloid β (N3pE) Assay Kit—IBL, Code No. 27716), which is to beused as reference and internal quality control. Capturing of the AβN3pE-40 peptide was done with the hAβ (x-40) ELISA (HS) from TGC (TheGenetics Company, Inc., Wagistrasse 23, 8952 Schlieren, Zurich areaSwitzerland), which should facilitate and expedite the process ofdevelopment.

SUMMARY OF THE INVENTION

The present invention pertains in particular to antibodies or variantsthereof, which are characterized in that they bind to Aβ-peptide with ahigh affinity. Said high affinity means in the context of the presentinvention an affinity of a K_(D) value of 10⁻⁷ M or better, preferably aK_(D) value of 10⁻⁸ M or better, and even more preferably a K_(D) valueof 10⁻⁹ M-10⁻¹² M.

In particular the antibody is preferably a monoclonal antibody and isselected from the following group

-   -   Aβ 5-5-6    -   Aβ 6-1-6    -   Aβ 17-4-3    -   Aβ 24-2-3

The antibody according to the present invention is especially useful ina diagnostic method to detect amyloidosis, in particular Alzheimer'sdisease.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE FIGURES

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1

A) Detection of 10 ng/ml amyloid D N3pE-40 by increasing concentrationsof pGlu-6166 antibody (clone 12-1).

B) Determination of the highest concentration of pGlu-6166 antibody(clone 12-1) required for detection of 10 ng/ml amyloid β N3pE-40.

FIG. 2

Dot Blot analysis of hybridoma cell culture supernatants of individualIgG producing clones.

FIG. 3

PepSpot Analysis of pGlu-6166 Hybridoma Cell Clones and IBL-Aβ N3pEantibody.

FIG. 4

12% SDS-PAGE of 20 μg pGlu-6166 antibody and hybridoma cell culturesupernatants.

FIG. 5

Biacore analysis of hybridoma cell culture supernatants. An overlay ofmonitored binding courses is illustrated graphically.

FIG. 6

Sensograms of interaction of anti-AβN3pE antibody clone 6-1-6 withAβpE3-40.

FIG. 7

Sensograms of interaction of anti-AβN3pE antibody clone 24-2-3 withAβpE3-40.

FIG. 8

N3pE-ELISA for clone 6-1-6, standard curve of AβpE3-40.

FIG. 9:

Sensograms of N3pE antibody clone 6-1-6.

FIG. 10

Quantification of AβpE3-42 using the method of neutralization by 1:20dilution in EIA buffer, pH titration with 860 μl 3.5 M Tris.

FIG. 11

Stained brain sections form Alzheimer's disease (AD) patients

(A) Brain of a sporadic AD (SAD) patient stained with anti-AP antibody6E10, recognizing total AP,(B) Brain of a sporadic AD (SAD) patient stained with N3pE antibodyclone 24-2-3, recognizing AβpE3-x,(C) Brain of a familial AD (FAD) patient stained with N3pE antibodyclone 24-2-3, recognizing AβpE3-x.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS Definitions

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The term “antibody” is used in the broadest sense and specificallycovers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity. The antibody may be an IgM, IgG(e.g. IgG₁, IgG2, IgG3 or IgG4), IgD, IgA or IgE, for example.Preferably however, the antibody is not an IgM antibody.

“Antibody fragments” comprise a portion of an intact antibody, generallythe antigen binding or variable region of the intact antibody. Examplesof antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments:diabodies; single-chain antibody molecules; and multispecific antibodiesformed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to “polyclonalantibody” preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies can frequentlybe advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKöhler et al., Nature, 256:495 (1975), or may be made by generally wellknown recombinant DNA methods. The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include chimericantibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain a minimal sequence derived from a non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences.

These modifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986), Reichmann et al, Nature. 332:323-329 (1988): andPresta, Curr. Op. Struct. Biel., 2:593-596 (1992). The humanizedantibody includes a Primatized™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(D)) in thesame polypeptide chain (V_(H)-V_(D)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in Hollinger et al., Proc. Natl. Acad. Sol. USA, 90:6444-6448(1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

As used herein, the expressions “cell”, “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and culture derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, this will be clearfrom the context.

The terms “polypeptide”, “peptide”, and “protein”, as used herein, areinterchangeable and are defined to mean a biomolecule composed of aminoacids linked by a peptide bond.

The terms “a”, “an” and “the” as used herein are defined to mean “one ormore” and include the plural unless the context is inappropriate.

The language “diseases and disorders which are caused by or associatedwith amyloid or amyloid-like proteins” includes, but is not limited to,diseases and disorders caused by the presence or activity ofamyloid-like proteins in monomeric, fibril, or polymeric state, or anycombination of the three. Such diseases and disorders include, but arenot limited to, amyloidosis, endocrine tumors, and macular degeneration.

The term “amyloidosis” refers to a group of diseases and disordersassociated with amyloid plaque formation including, but not limited to,secondary amyloidosis and age-related amyloidosis such as diseasesincluding, but not limited to, neurological disorders such asAlzheimer's Disease (AD), including diseases or conditions characterizedby a loss of cognitive memory capacity such as, for example, mildcognitive impairment (MCI), sporadic Alzheimer's disease, Lewy bodydementia, Down's syndrome, hereditary cerebral hemorrhage withamyloidosis (Dutch type); the Guam Parkinson-Dementia complex, familialforms of Alzheimer's disease like Familial British Dementia (FBD) andFamilial Danish Dementia (FDD); as well as other diseases which arebased on or associated with amyloid-like proteins such as progressivesupranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,Parkinson's disease, HIV-related dementia, ALS (amyotropic lateralsclerosis), inclusion-body myositis (IBM), Adult Onset Diabetes, andsenile cardiac amyloidosis; and various eye diseases including maculardegeneration, drusen-related optic neuropathy, and cataract due tobeta-amyloid deposition.

“Amyloid β, Aβ or /β-amyloid” is an art recognized term and refers toamyloid β proteins and peptides, amyloid β precursor protein (APP), aswell as modifications, fragments and any functional equivalents thereof.In particular, by amyloid β as used herein is meant any fragmentproduced by proteolytic cleavage of APP but especially those fragmentswhich are involved in or associated with the amyloid pathologiesincluding, but not limited to, Aβ₁₋₃₈, Aβ₁₋₄₀, Aβ₁₋₄₂. The amino acidsequences of these Aβ peptides are as follows:

Aβ 1-42 (SEQ ID NO. 1): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly-Val-Val-Ile-AlaAβ 1-40 (SEQ ID NO. 2): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly-Val-Val Aβ 1-38(SEQ ID NO. 3): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly

“pGlu-Aβ” or “Aβ N3pE” refers to N-terminally truncated forms of AP,that start at the glutamic acid residue at position 3 in the amino acidsequence of Aβ, and wherein said glutamic acid residue is cyclized toform a pyroglutamic acid residue. In particular, by pGlu-Aβ as usedherein are meant those fragments which are involved in or associatedwith the amyloid pathologies including, but not limited to, pGlu-Aβ₃₋₃₈,pGlu-Aβ₃₋₄₀, p-Glu-Aβ₃₋₄₂.

The sequences of the N-terminally truncated forms of Aβ, Aβ₃₋₃₈, Aβ₃₋₄₀,Aβ₃₋₄₂ are as follows:

Aβ 3-42 (SEQ ID NO. 4): Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly- Val-Val-Ile-Ala Aβ 3-40(SEQ ID NO. 5): Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly- Val-Val Aβ 3-38 (SEQ IDNO. 6): Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-GlyIn particular the present invention pertains to the following items:

-   1. Antibody, characterised in that it binds to Aβ peptides or    variants thereof, preferably with high affinity.-   2. Antibody according to item 1, wherein said high affinity means a    dissociation constant (K_(D)) value of 10⁻⁷ M, or better.-   3. Antibody according to item 1 or 2, wherein said antibody is a    monoclonal antibody.-   4. Antibody according to any of the preceding items, wherein the    variable part of the light chain of said antibody has a nucleotide    sequence selected from SEQ ID NOs: 49, 53, 57 and 61, or having an    amino acid sequence selected from SEQ ID NOs: 50, 54, 58, and 62.-   5. Antibody according to any of the preceding items, wherein the    variable part of the heavy chain of said antibody has a nucleotide    sequence selected from SEQ ID NOs: 51, 55, 59 and 63, or having an    amino acid sequence selected from SEQ ID NOs: 52, 56, 60 and 64.-   6. Antibody according to any of the preceding items, wherein the    variable part of the light chain of said antibody has the nucleotide    sequence of SEQ ID NO: 49 or the amino acid sequence of SEQ ID NO:    50, and wherein the variable part of the heavy chain of said    antibody has the nucleotide sequence of SEQ ID NO: 51, or the amino    acid sequence of SEQ ID NO: 52.-   7. Antibody according to any of the preceding items, wherein the    variable part of the light chain of said antibody has the nucleotide    sequence of SEQ ID NO: 53 or the amino acid sequence of SEQ ID NO:    54, and wherein the variable part of the heavy chain of said    antibody has the nucleotide sequence of SEQ ID NO: 55, or the amino    acid sequence of SEQ ID NO: 56.-   8. Antibody according to any of the preceding items, wherein the    variable part of the light chain of said antibody has the nucleotide    sequence of SEQ ID NO: 57 or the amino acid sequence of SEQ ID NO:    58, and wherein the variable part of the heavy chain of said    antibody has the nucleotide sequence of SEQ ID NO: 59, or the amino    acid sequence of SEQ ID NO: 60.-   9. Antibody according to any of the preceding items, wherein the    variable part of the light chain of said antibody has the nucleotide    sequence of SEQ ID NO: 61 or the amino acid sequence of SEQ ID NO:    62, and wherein the variable part of the heavy chain of said    antibody has the nucleotide sequence of SEQ ID NO: 63, or the amino    acid sequence of SEQ ID NO: 64.-   10. Antibody according to any of the preceding items, wherein said    antibody is selected from the following group:

Aβ 5-5-6 (Deposit No. DSM ACC 2923) Aβ 6-1-6 (Deposit No. DSM ACC 2924)Aβ 17-4-3 (Deposit No. DSM ACC 2925) Aβ 24-2-3 (Deposit No. DSM ACC2926)

-   -   or functional variants thereof.

-   11. Antibody according to any of the preceding items, wherein said    antibody is Aβ 6-1-6 (Deposit No. DSM ACC 2924).

-   12. Antibody according to any of the preceding items, wherein said    antibody is Aβ 24-2-3 (Deposit No. DSM ACC 2926).

-   13. Antibody according to any of the preceding items, wherein said    antibody is a humanized or chimeric antibody, or an antibody    fragment which retains the high affinity.

-   14. Antibody according to any of the preceding items for use in the    detection of Aβ peptide or variants thereof.

-   15. Antibody according to item 14, wherein said variants are    selected from the following group:    -   pGlu-Aβ₃₋₃₈    -   pGlu-Aβ₃₋₄₀    -   pGlu-Aβ₃₋₄₂, and    -   pGlu-Aβ_(3-x), variants,    -   wherein x is an integer between 10 and 42; preferably 18 and 42,        more preferably 30 and 42.

-   16. Antibody according to any of the preceding items, which is    human.

-   17. Antibody according to any of the preceding items, which is a    diabody or a single chain antibody which retains the high affinity.

-   18. Antibody according to any of the preceding items, which binds to    the epitope bound by the antibodies defined in item 15.

-   19. Antibody according to any of the preceding items, which has the    complementarity determining regions of the antibodies as defined in    item 15.

-   20. Antibody according to any of the preceding items, which is    labeled.

-   21. Antibody according to any of the preceding items, which is    immobilised on a solid phase.

-   22. Antibody obtainable from any one of hybridoma cell lines DSM ACC    2923, DSM ACC 2924, DSM ACC 2925, DSM ACC 2926.

-   23. Composition comprising the antibody as defined in any of the    preceding items.

-   24. Composition according to item 23 for the treatment, prevention    or delay of amyloidosis.

-   25. Composition according to item 23 or 24, wherein said amyloidosis    is a neurodegenerative disease selected from the group consisting of    mild cognitive impairment, Alzheimer's disease and neurodegeneration    in Down Syndrome.

-   26. Composition according to item 23 or 24, wherein said amyloidosis    is sporadic Alzheimer's disease or a Familial Alzheimer's dementia.

-   27. Composition according to item 26, wherein said Familial    Alzheimer's dementia is Familial British Dementia or Familial Danish    Dementia.

-   28. Hybridoma cell line DSM ACC 2923.

-   29. Hybridoma cell line DSM ACC 2924

-   30. Hybridoma cell line DSM ACC 2925.

-   31. Hybridoma cell line DSM ACC 2926.

-   32. Use of the antibody as defined in any one of items 1 to 22 or    the composition as defined in any one of items 23 to 27 in a    diagnostic or therapeutic method.

-   33. The use according to item 32 for the diagnosis of an    amyloid-associated disease or condition.

-   34. The use according to item 33, wherein said amyloidosis is a    neurodegenerative disease selected from the group consisting of mild    cognitive impairment, Alzheimer's disease and neurodegeneration in    Down Syndrome.

-   35. The use according to item 33, wherein said amyloidosis is    sporadic Alzheimer's disease or a Familial Alzheimer's dementia.

-   36. The use according to item 35, wherein said Familial Alzheimer's    dementia is Familial British Dementia or Familial Danish Dementia.

-   37. In vitro diagnostic method for the diagnosis of an    amyloid-associated disease or condition, in particular Alzheimer's    disease, comprising the following steps:    -   contacting an antibody according to any one of items 1 to 22        with a sample from a subject suspected to be afflicted with said        disease or condition, and    -   detecting binding of the antibody to a pGlu-amyloid protein,        preferably pGlu-Aβ peptide from the sample.

-   38. Diagnostic kit, comprising the antibody as defined in any one of    items 1 to 22, and instructions for use, and —optionally—(a) further    biologically active substance(s).

-   39. The diagnostic kit of item 32, wherein said further biological    substance is an inhibitor of glutaminy cyclase.

-   40. An oligonucleotide selected from the group consisting of SEQ ID    Nos: 23 to 48.

The antibodies of the invention may be useful for the diagnosis ofamyloidosis.

The antibodies of the invention may be used as affinity purificationagents. In this process, the antibodies are immobilised on a solid phasesuch a Sephadex resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theAβ-peptide (or fragment thereof) to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the Aβ-peptide, which is bound tothe immobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0 that will release theAβ-peptide from the antibody.

Anti-Aβ-peptide antibodies may also be useful in diagnostic assays forAβ-peptide, e.g. detecting its occurrence in specific cells, tissues, orserum. Thus, the antibodies may be used in the diagnosis of amyloidosis,in particular a neurodegenerative disease selected from the groupconsisting of mild cognitive impairment (MCI), Alzheimer's disease (AD),like for instance sporadic Alzheimer's disease (SAD) or FamilialAlzheimer's dementias (FAD) such as Familial British Dementia (FBD) andFamilial Danish Dementia (FDD) and neurodegeneration in Down Syndrome;preferably Alzheimer's disease.

For diagnostic applications, the antibody typically will be labelledwith a detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Gütigen et al., Ed.,Wiley-Interscience. New York, N.Y. Pubs., (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available. The enzyme generallycatalyses a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase.0-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym (ed Langone & H. Van Vunakis),Academic Press, New York, 73: 147-166 (1981).

Examples of enzyme-substrate combinations include, for example(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g. orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.p-nitrophenyl-β-D-galactosidase) or the fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g. digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g. anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

The Aβ-antibody needs not be labeled, and the presence thereof can bedetected using a labeled antibody, which binds to the Aβ-antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies A Manual of Techniques, pp. 147-158 (CRC Press. Inc., 1987)

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of Aβ peptide in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. The second antibody may itself be labeled with adetectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one preferable type of sandwichassay is an ELISA assay, in which case the detectable moiety is anenzyme.

For immunohistochemistry, the tissue sample may be fresh or frozen ormay be embedded in paraffin and fixed with a preservative such asformalin, for example.

Diagnostic Kits

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labelled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g. asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g. a block buffer or lysis buffer) and the like.The relative amounts of the various reagents may be varied widely toprovide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

The diagnostic kit according to the invention may contain a furtherbiologically active substance as described below. Especially preferredfor the use in the diagnostic kit are inhibitors of glutaminyl cyclase.

The diagnostic kit of the invention is especially useful for thedetection and diagnosis of amyloid-associated diseases and conditions,in particular neurodegenerative diseases selected from the groupconsisting of mild cognitive impairment (MCI), Alzheimer's disease (AD),like for instance sporadic Alzheimer's disease (SAD) or FamilialAlzheimer's dementias (FAD) like Familial British Dementia (FBD) andFamilial Danish Dementia (FDD), neurodegeneration in Down Syndrome;preferably Alzheimer's disease.

The present invention pertains in particular to antibodies which arecharacterized in that they bind to Aβ-peptide with a high affinity. Thepresent invention also pertains to antibodies which are characterised inthat they bind to Aβ-peptides or variants thereof with a high affinity.Said high affinity means in the context of the present invention anaffinity of a K_(D) value of 10⁻⁷ M or better, preferably a K_(D) valueof 10⁻⁸ M or better, and even more preferably a K_(D) value of 10⁻⁹M-10⁻¹² M. Thereby, the inventive antibodies bind to Aβ-peptide with ahigher affinity than previously known antibodies.

In particular the antibody is preferably a monoclonal antibody and isselected from the following group

Aβ 5-5-6 (DSM ACC 2923) Aβ 6-1-6 (DSM ACC 2924) Aβ 17-4-3 (DSM ACC 2925)Aβ 24-2-3 (DSM ACC 2926)

The antibody according to the present invention is especially useful ina diagnostic method to detect amyloidosis, in particular aneurodegenerative disease selected from the group consisting of mildcognitive impairment (MCI), Alzheimer's disease (AD), like for instancesporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias(FAD) like Familial British Dementia (FBD) and Familial Danish Dementia(FDD), neurodegeneration in Down Syndrome; preferably Alzheimer'sdisease.

According to a preferred embodiment, the antibody can be humanised or isa chimeric antibody or is a human antibody.

Further, the antibody as selected from the above-mentioned group canalso be a functional variant of said group.

In the context of the present invention, a variant of an p-Glu-Aβpeptide is in particular

-   -   pGlu-Aβ₃₋₃₈,    -   pGlu-Aβ₃₋₄₀,    -   pGlu-Aβ₃₋₄₂

Further variants of Aβ peptides are all pGlu-Aβ_(3-x) variants, whichhave been shown to accumulate in the brain as a consequence ofAlzheimer's disease or preceding Alzheimer's disease. X is defined as aninteger between 10 and 42, e.g. in the above pGlu-Aβ₃₋₄₂, “42” would bethe integer for “x”.

In the context of the present invention a “functional variant” of theinventive antibody is an antibody which retains the binding capacities,in particular binding capacities with high affinity to a pGlu-Aβ_(3-x)peptide or functional variant thereof. The provision of such functionalvariants is known in the art and encompasses the above-mentionedpossibilities, which were indicated under the definition of antibodiesand fragments thereof.

In a preferred embodiment, the antibody is an antibody fragment, asdefined above.

In a further preferred embodiment, the inventive antibody is an antibodywhich binds to the epitope which is bound by the antibodies as definedabove, in particular antibody 5-5-6, antibody 6-1-6, antibody 17-4-3 andantibody 24-2-3.

In a further preferred embodiment, the antibody of the invention is anantibody which has the complementarity-determining regions (CDRs) of theabove-defined antibodies. Preferably, the antibody can be labeled;possible labels are those as mentioned above and all those known to aperson skilled in the art of diagnostic uses of antibodies inparticular.

Preferably, the antibody is immobilized on a solid phase.

The present invention also concerns an antibody which is obtainable fromhybridoma cell line 6-1-6 (DSM ACC 2924).

The present invention also relates to a composition which comprises theantibody as defined above. In particular, said composition is acomposition for a diagnostic use, especially for the diagnosis of aneurodegenerative disease selected from the group consisting of mildcognitive impairment (MCI), Alzheimer's disease (AD), like for instancesporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias(FAD) like Familial British Dementia (FBD) and Familial Danish Dementia(FDD), neurodegeneration in Down Syndrome; preferably Alzheimer'sdisease; in particular by detection of Aβ peptide or variants thereof ina biological sample.

In another embodiment, the antibody according to the invention and asdescribed herein before or a fragment thereof, exhibits an bindingaffinity to an Aβ oligomer, fiber, fibril or filament which is at least2 times, particularly at least 4 times, particularly at least 10 times,particularly at least 15 times, more particularly at least 20 times, butespecially at least 25 times higher than the binding affinity to an Aβmonomer.

In still another embodiment, an antibody or a fragment thereof or achimeric antibody or a fragment thereof, or a humanized antibody or afragment thereof is provided as described herein before, which antibodysubstantially binds to aggregated Aβ, including Aβ plaques, in themammalian, particularly the human brain but, preferably, does not showany significant cross-reactivity with amyloid precursor protein (APP).

In another aspect of the invention, the antibody or a fragment thereofor the chimeric antibody or a fragment thereof, or a humanized antibodyor a fragment thereof is provided as described herein before, whichantibody substantially binds to soluble polymeric amyloid, particularlyamyloid β (Aβ), including Aβ monomers, in the mammalian, particularlythe human brain but, preferably, does not show any significantcross-reactivity with amyloid precursor protein (APP).

The present invention relates also to humanized forms of the antibodiesas defined above, compositions comprising said humanized antibodies andthe use of said compositions for the treatment of amyloidosis,especially for the treatment of neurodegenerative disease in a mammal,in particular in a human. Said neurodegenerative disease is inparticular selected from the group consisting of mild cognitiveimpairment (MCI), Alzheimer's disease (AD), like for instance sporadicAlzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) likeFamilial British Dementia (FBD) and Familial Danish Dementia (FDD),neurodegeneration in Down Syndrome. Preferably, said neurodegenerativedisease is Alzheimer's disease.

The present invention is also directed to the following hybridoma celllines 5-5-6, 6-1-6, 17-4-3 and 24-2-3.

The present invention also pertains to the use of the antibody or thecomposition comprising the antibody, both as defined above, for use inan in vitro diagnostic method. In particular, this diagnostic method isdirected to diagnosis of a neurodegenerative disease selected from thegroup consisting of mild cognitive impairment (MCI), Alzheimer's disease(AD), like for instance sporadic Alzheimer's disease (SAD) or FamilialAlzheimer's dementias (FAD) like Familial British Dementia (FBD) andFamilial Danish Dementia (FDD), neurodegeneration in Down Syndrome;preferably Alzheimer's disease; especially by detecting an Aβ peptide orvariants thereof in a biological sample.

Preferably, said sample is a serum sample.

According to another preferred embodiment, said sample is a liquor orcerebrospinal fluid (CSF) sample.

In a particularly preferred embodiment, the present invention pertainsto the following method:

In vitro or in situ diagnostic method for the diagnosis of anamyloid-associated disease or condition, preferably Alzheimer's disease,comprising the following steps:

contacting an antibody according to the invention with a sample,preferably selected from a serum, liquor or CSF sample, most preferablya serum sample; or a specific body part or body area of a subjectsuspected to be afflicted with said condition or disease, anddetecting binding of the antibody to a pGlu-amyloid protein, preferablypGlu-Aβ peptide, from the sample.

More particularly, the invention relates to a method of diagnosis of anamyloid-associated disease or condition, preferably Alzheimer's disease,comprising detecting the immunospecific binding of an antibody or anactive fragment thereof to a pGlu-amyloid protein, preferably pGlu-Aβpeptide, in a sample or in situ which includes the steps of

(a) bringing the sample or a specific body part or body area suspectedto contain the amyloid protein into contact with an antibody,particularly a monoclonal antibody according to the invention, or achimeric antibody or a fragment thereof, or a humanized antibody or afragment thereof according to the invention and as described hereinbefore, and/or a functional part thereof, which antibody binds a pGlu-Appeptide;(b) allowing the antibody and/or a functional part thereof, to bind tothe pGlu-Aβ peptide to form an immunological complex;(c) detecting the formation of the immunological complex; and(d) correlating the presence or absence of the immunological complexwith the presence or absence of pGlu-Aβ peptide in the sample orspecific body part or area.

Also comprised is a method of determining the extent of amyloidogenicplaque burden in a tissue and/or body fluids comprising

(a) obtaining a sample representative of the tissue and/or body fluidsunder investigation;(b) testing said sample for the presence of amyloid protein with anantibody, particularly a monoclonal antibody according to the invention,or a chimeric antibody or a fragment thereof, or a humanized antibody ora fragment thereof according to the invention and as described hereinbefore, and/or a functional part thereof;(c) determining the amount of antibody bound to the protein; and(d) calculating the plaque burden in the tissue and/or body fluids.

In particular, the invention relates to a method of determining theextent of amyloidogenic plaque burden in a tissue and/or body fluids,wherein the formation of the immunological complex in step c) isdetermined such that presence or absence of the immunological complexcorrelates with presence or absence of amyloid protein, in particularpGlu-Aβ peptides.

In still another embodiment, the invention relates to a compositioncomprising the antibody according to the invention, or a chimericantibody or a fragment thereof, or a humanized antibody or a fragmentthereof according to the invention and as described herein beforeincluding any functionally equivalent antibody or any derivative orfunctional parts thereof, in a therapeutically effective amount, inparticular a composition which is a pharmaceutical compositionoptionally further comprising a pharmaceutically acceptable carrier.

In another embodiment of the invention, said composition comprises theantibody in a therapeutically effective amount. Further comprised by theinvention is a mixture comprising an antibody, particularly a monoclonalantibody according to the invention, or a chimeric antibody or afragment thereof, or a humanized antibody or a fragment thereofaccording to the invention and as described herein before including anyfunctionally equivalent antibody or any derivative or functional partsthereof, in a therapeutically effective amount and, optionally, afurther biologically active substance and/or a pharmaceuticallyacceptable carrier and/or a diluent and/or an excipient.

In particular, the invention relates to a mixture, wherein the furtherbiologically active substance is a compound used in the medication ofamyloidosis, a group of diseases and disorders associated with amyloidor amyloid-like protein such as the Aβ protein involved inneurodegenerative diseases selected from the group consisting of mildcognitive impairment (MCI), Alzheimer's disease (AD), like for instancesporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias(FAD) like Familial British Dementia (FBD) and Familial Danish Dementia(FDD), neurodegeneration in Down Syndrome; preferably Alzheimer'sdisease.

In another embodiment of the invention, the other biologically activesubstance or compound may also be a therapeutic agent that may be usedin the treatment of amyloidosis caused by amyloid β or may be used inthe medication of other neurological disorders.

The other biologically active substance or compound may exert itsbiological effect by the same or a similar mechanism as the antibodyaccording to the invention or by an unrelated mechanism of action or bya multiplicity of related and/or unrelated mechanisms of action.

Generally, the other biologically active compound may includeneutron-transmission enhancers, psychotherapeutic drugs, acetylcholineesterase inhibitors, calcium-channel blockers, biogenic amines,benzodiazepine tranquillizers, acetylcholine synthesis, storage orrelease enhancers, acetylcholine postsynaptic receptor agonists,monoamine oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamatereceptor antagonists, non-steroidal anti-inflammatory drugs,antioxidants, and serotonergic receptor antagonists.

More particularly, the invention relates to a mixture comprising atleast one compound selected from the group consisting of compoundseffective against oxidative stress, anti-apoptotic compounds, metalchelators, inhibitors of DNA repair such as pirenzepin and metabolites,3-amino-1-propanesulfonic acid (3 APS), 1,3-propanedisulfonate (1,3PDS),α-secretase activators, β- and γ-secretase inhibitors, tau proteins,neurotransmitter, /3-sheet breakers, attractants for amyloid betaclearing /depleting cellular components, inhibitors of N-terminaltruncated amyloid beta including pyroglutamated amyloid beta 3-42, suchas inhibitors of glutaminyl cyclase, anti-inflammatory molecules, orcholinesterase inhibitors (ChEIs) such as tacrine, rivastigmine,donepezil, and/or galantamine, Ml agonists and other drugs including anyamyloid or tau modifying drug and nutritive supplements, and nutritivesupplements, together with an antibody according to the presentinvention and, optionally, a pharmaceutically acceptable carrier and/ora diluent and/or an excipient.

The invention further relates to a mixture, wherein the compound is acholinesterase inhibitor (ChEIs), particularly a mixture, wherein thecompound is one selected from the group consisting of tacrine,rivastigmine, donepezil, galantamine, niacin and memantine.

In a further embodiment, the mixtures according to the invention maycomprise niacin or memantine together with an antibody according to thepresent invention and, optionally, a pharmaceutically acceptable carrierand/or a diluent and/or an excipient.

In a further embodiment, the mixtures according to the invention maycomprise a glutaminyl cyclase inhibitor together with an antibodyaccording to the present invention and, optionally, a pharmaceuticallyacceptable carrier and/or a diluent and/or an excipient.

Preferred inhibitors of glutaminyl cyclase are described in WO2005/075436, in particular examples 1-141 as shown on pp. 31-40. Thesynthesis of examples 1-141 is shown on pp. 40-48 of WO 2005/075436. Thedisclosure of WO 2005/075436 regarding examples 1-141, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/055945, in particular examples 1-473 as shown on pp. 46-155. Thesynthesis of examples 1-473 is shown on pp. 156-192 of WO 2008/055945.The disclosure of WO 2008/055945 regarding examples 1-473, theirsynthesis and their use as glutaminyl cyclase inhibitors is incorporatedherein by reference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/055947, in particular examples 1-345 as shown on pp. 53-118. Thesynthesis of examples 1-345 is shown on pp. 119-133 of WO 2008/055947.The disclosure of WO 2008/055947 regarding examples 1-345, theirsynthesis and their use as glutaminyl cyclase inhibitors is incorporatedherein by reference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/055950, in particular examples 1-212 as shown on pp. 57-120. Thesynthesis of examples 1-212 is shown on pp. 121-128 of WO 2008/055950.The disclosure of WO 2008/055950 regarding examples 1-212, theirsynthesis and their use as glutaminyl cyclase inhibitors is incorporatedherein by reference.

Further preferred inhibitors of glutaminyl cyclase are described inWO2008/065141, in particular examples 1-25 as shown on pp. 56-59. Thesynthesis of examples 1-25 is shown on pp. 60-67 of WO2008/065141. Thedisclosure of WO2008/065141 regarding examples 1-25, their synthesis andtheir use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/110523, in particular examples 1-27 as shown on pp. 55-59. Thesynthesis of examples 1-27 is shown on pp. 59-71 of WO 2008/110523. Thedisclosure of WO 2008/110523 regarding examples 1-27, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128981, in particular examples 1-18 as shown on pp. 62-65. Thesynthesis of examples 1-18 is shown on pp. 65-74 of WO 2008/128981. Thedisclosure of WO 2008/128981 regarding examples 1-18, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128982, in particular examples 1-44 as shown on pp. 61-67. Thesynthesis of examples 1-44 is shown on pp. 68-83 of WO 2008/128982. Thedisclosure of WO 2008/128982 regarding examples 1-44, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128983, in particular examples 1-30 as shown on pp. 64-68. Thesynthesis of examples 1-30 is shown on pp. 68-80 of WO 2008/128983. Thedisclosure of WO 2008/128983 regarding examples 1-30, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128984, in particular examples 1-36 as shown on pp. 63-69. Thesynthesis of examples 1-36 is shown on pp. 69-81 of WO 2008/128984. Thedisclosure of WO 2008/128984 regarding examples 1-36, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128985, in particular examples 1-71 as shown on pp. 66-76. Thesynthesis of examples 1-71 is shown on pp. 76-98 of WO 2008/128985. Thedisclosure of WO 2008/128985 regarding examples 1-71, their synthesisand their use as glutaminyl cyclase inhibitors is incorporated herein byreference.

Further preferred inhibitors of glutaminyl cyclase are described in WO2008/128986, in particular examples 1-7 as shown on pp. 65-66. Thesynthesis of examples 1-7 is shown on pp. 66-73 of WO 2008/128986. Thedisclosure of WO 2008/128986 regarding examples 1-7, their synthesis andtheir use as glutaminyl cyclase inhibitors is incorporated herein byreference.

In still another embodiment of the invention mixtures are provided thatcomprise “atypical antipsychotics” such as, for example clozapine,ziprasidone, risperidone, aripiprazole or olanzapine for the treatmentof positive and negative psychotic symptoms including hallucinations,delusions, thought disorders (manifested by marked incoherence,derailment, tangentiality), and bizarre or disorganized behavior, aswell as anhedonia, flattened affect, apathy, and social withdrawal,together with an antibody, particularly a monoclonal antibody accordingto the invention, but particularly a chimeric antibody or a fragmentthereof, or a humanized antibody or a fragment thereof according to theinvention and as described herein and, optionally, a pharmaceuticallyacceptable carrier and/or a diluent and/or an excipient.

In a specific embodiment of the invention, the compositions and mixturesaccording to the invention and as described herein before comprise theantibody and the biologically active substance, respectively, in atherapeutically effective amount.

Other compounds that can be suitably used in mixtures in combinationwith the antibody according to the present invention are described inWO2008/065141 (see especially pages 37/38), including PEP-inhibitors(pp. 43/44), LiCl, inhibitors of dipeptidyl aminopeptidases, preferablyinhibitors of DP IV or DP IV-like enzymes (see pp. 48/49);acetylcholinesterase (ACE) inhibitors (see p. 47), PIMT enhancers,inhibitors of beta secretases (see p. 41), inhibitors of gammasecretases (see pp. 41/42), inhibitors of neutral endopeptidase,inhibitors of phosphodiesterase-4 (PDE-4) (see pp. 42/43), TNFalphainhibitors, muscarinic M1 receptor antagonists (see p. 46), NMDAreceptor antagonists (see pp. 47/48), sigma-1 receptor inhibitors,histamine H3 antagonists (se p. 43), immunomodulatory agents,immunosuppressive agents or an agent selected from the group consistingof antegren (natalizumab), Neurelan (fampridine-SR), campath(alemtuzumab), IR 208, NBI 5788/MSP 771 (tiplimotide), paclitaxel,Anergix.MS (AG 284), SH636, Differin (CD 271, adapalene), BAY 361677(interleukin-4), matrix-metalloproteinase-inhibitors (e.g. BB 76163),interferon-tau (trophoblastin) and SAIK-MS; beta-amyloid antibodies (seep. 44), cysteine protease inhibitors (see p. 44); MCP-1 antagonists (seepp. 44/45), amyloid protein deposition inhibitors (see 42) and betaamyloid synthesis inhibitors (see p. 42), which document is incorporatedherein by reference.

In another embodiment, the invention relates to a mixture comprising theantibody, particularly a monoclonal antibody according to the invention,or a chimeric antibody or a fragment thereof, or a humanized antibody ora fragment thereof according to the invention and as described hereinbefore and/or the biologically active substance in a therapeuticallyeffective amount.

The invention further relates to the use of an antibody, particularly amonoclonal antibody according to the invention, but particularly achimeric antibody or a fragment thereof, or a humanized antibody or afragment thereof according to the invention and as described hereinbefore and/or a functional part thereof and/or a pharmaceuticalcomposition, or a mixture comprising said antibody, for the preparationof a medicament for treating or alleviating the effects of amyloidosis,a group of diseases and disorders associated with amyloid plaqueformation including secondary amyloidosis and age-related amyloidosissuch as diseases including, but not limited to, neurological disorderssuch as Alzheimer's Disease (AD), Lewy body dementia, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex; as well as other diseases which are based onor associated with amyloid-like proteins such as progressivesupranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,Parkinson's disease, HIV-related dementia, ALS (amyotropic lateralsclerosis), Adult Onset Diabetes; senile cardiac amyloidosis; endocrinetumors, and others, including macular degeneration.

Also comprised by the present invention is a method for the preparationof an antibody, particularly a monoclonal antibody according to theinvention, but particularly a chimeric antibody or a fragment thereof,or a humanized antibody or a fragment thereof according to the inventionand as described herein before and/or a functional part thereof and/or apharmaceutical composition, or a mixture comprising said antibody and/ora functional part thereof, particularly in a therapeutically effectiveamount, for use in a method of preventing, treating or alleviating theeffects of amyloidosis, a group of diseases and disorders associatedwith amyloid plaque formation including secondary amyloidosis andage-related amyloidosis such as diseases including, but not limitedtoneurodegenerative diseases such as mild cognitive impairment (MCI),Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease(SAD) or Familial Alzheimer's dementias (FAD) like Familial BritishDementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration inDown Syndrome; Lewy body dementia, hereditary cerebral hemorrhage withamyloidosis (Dutch type); the Guam Parkinson-Dementia complex; as wellas other diseases which are based on or associated with amyloid-likeproteins such as progressive supranuclear palsy, multiple sclerosis;Creutzfeld Jacob disease, Parkinson's disease, HIV-related dementia, ALS(amyotropic lateral sclerosis), Adult Onset Diabetes; senile cardiacamyloidosis; endocrine tumors, and others, including maculardegeneration comprising formulating an antibody, particularly amonoclonal antibody according to the invention, but particularly achimeric antibody or a fragment thereof, or a humanized antibody or afragment thereof according to the invention in a pharmaceuticallyacceptable form.

Further comprised by the present invention is a method for preventing,treating or alleviating the effects of amyloidosis, a group of diseasesand disorders associated with amyloid plaque formation includingsecondary amyloidosis and age-related amyloidosis such as diseasesincluding, but not limited to, neurological disorders such as mildcognitive impairment (MCI), Alzheimer's disease (AD), like for instancesporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias(FAD) like Familial British Dementia (FBD) and Familial Danish Dementia(FDD), neurodegeneration in Down Syndrome; Lewy body dementia,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex; as well as other diseases which are based onor associated with amyloid-like proteins such as progressivesupranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,Parkinson's disease, HIV-related dementia, ALS (amyotropic lateralsclerosis), Adult Onset Diabetes; senile cardiac amyloidosis; endocrinetumors, and others, including macular degeneration by administering anantibody and/or a functional part thereof, but particularly a humanizedantibody and/or a functional part thereof, or a composition or mixturecomprising such an antibody and/or a functional part thereof, to ananimal or a human affected by such a disorder comprising administeringthe antibody in a therapeutically effective amount.

It is also an object of the invention to provide a method for thetreatment of amyloidosis, a group of diseases and disorders associatedwith amyloid plaque formation including secondary amyloidosis andage-related amyloidosis including, but not limited to, neurodegenerativediseases such as mild cognitive impairment (MCI), Alzheimer's disease(AD), like for instance sporadic Alzheimer's disease (SAD) or FamilialAlzheimer's dementias (FAD) like Familial British Dementia (FBD) andFamilial Danish Dementia (FDD), neurodegeneration in Down Syndrome;particularly a disease or condition characterized by a loss of cognitivememory capacity by administering to an animal, particularly a mammal ora human, an antibody, particularly a pharmaceutical compositionaccording to the invention and as described herein.

In a specific embodiment the invention provides a method for retainingor increasing cognitive memory capacity but, particularly, for restoringthe cognitive memory capacity of an animal, particularly a mammal or ahuman, suffering from memory impairment by administering to an animal,particularly a mammal or a human, an antibody, particularly apharmaceutical composition according to the invention and as describedherein before.

It is a further object of the invention to provide a therapeuticcomposition and a method of producing such a composition as well as amethod for the treatment of amyloidosis, a group of diseases anddisorders associated with amyloid plaque formation including secondaryamyloidosis and age-related amyloidosis including, but not limited to,neurodegenerative diseases such as mild cognitive impairment (MCI),Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease(SAD) or Familial Alzheimer's dementias (FAD) like Familial BritishDementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration inDown Syndrome; particularly a disease or condition characterized by aloss of cognitive memory capacity, using an antibody according to theinvention and as described herein before.

In particular, the invention relates to the treatment of an animal,particularly a mammal or a human, suffering from an amyloid-associatedcondition characterized by a loss of cognitive memory capacity thatleads to the retention of cognitive memory capacity.

Having described the invention in detail, it will be apparent thatmodifications, variations, and equivalent embodiments are possiblewithout departing the scope of the invention defined in the appendedclaims. Furthermore, it should be appreciated that all examples in thepresent disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present invention. It should be appreciated by those of skill in theart that the techniques disclosed in the examples that follow representapproaches the inventors have found function well in the practice of theinvention, and thus can be considered to constitute examples of modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

1. Material and Methods 1.1 Production of Antibodies Mice

For the production of hybridomas, female BALB/C mice (Charles River) of8 weeks age were used.

Myeloma Cell Line

For the generation of the hybridomas, the myeloma cell line SP2/0-Ag14from Deutsche Stammsammlung von Mikoorganismen und Zellkulturen wasused.

Antigen

The peptide pGlu-6166 (sequence pGlu-FRHDSGC, SEQ ID NO: 65) was used.

Strategy

As an immunogen, the peptide was coupled to bovine thyroglubulin (BTG,SIGMA) via maleimid groups from three different linkers. The threelinkers of different length were used fromN-[e-maleimidocaproyloxy]succinimide ester (EMCS),succiminidyl-4-(N-maleimidomethyl)-cyclohexan-1-carboxy-(6-amidocaproate)(LCSMCC) and N-[b-maleimidopropyloxy]succinimide ester (BMPS).

For the detection of the generated antibodies, the same peptide wasconjugated to bovine serum albumine (BSA, SIGMA) via maleimid groupsfrom succinimidyl-6-[(b-maleimido-propionamido)hexanoate] (SMPH).

Methods Conjugation of the Peptide for Immunization

Conjugation was performed in two steps via SH-groups from the cysteinresidue of the peptide.

1. Maleoylation of the Carrier Protein

2 to 5 mg of the respective linker (50 mg/ml in N-methylpyrrolidone,NMP) was added to 2 ml of the carrier protein solution (10 mg/ml in 0.1mM NaHCO₃, pH 8.0). The reaction mixture was incubated for 1 h at roomtemperature (RT). The reaction mixture was thereafter desalted using aSephadex G-50 column (1.5×14 cm), which was equilibrated with 50 mMsodium phosphate, 250 mM NaCl, pH 6.8.

2. Coupling of the Maleoylated BTG with the Peptide

250 μl of the peptide solution (10 mg/ml in Aqua bidest) were mixed with2 ml of a solution containing the maleoylated carrier proteins (2.5mg/ml) in 50 mM sodium phosphate, 250 ml NaCl, pH 6.8 and incubated for2 h at 4° C. and further 4 h at RT. Unreacted maleimid groups wereblocked by addition of 2-mercaptoethanole up to a concentration of 10 mMand over night incubation at 4° C. The resulting conjugate was dialysedagainst 10 mM sodium phosphate, 150 mM NaCl, pH 7.5 at 4° C. (3 timesbuffer exchange, MW cut-off 10.000).

Conjugation of the Peptide for ELISA

Conjugation was performed in two steps via SH-groups from the cysteinresidue of the peptide.

1. Maleoylation of the Carrier Protein

2 mg SMPH (50 mg/ml in N-methylpyrrolidone, NMP) was added to 2 ml ofthe carrier protein solution (BSA, 10 mg/ml in 0.1 mM NaHCO₃, pH 8.0).The reaction mixture was incubated for 1 h at room temperature (RT). Thereaction mixture was thereafter desalted using a Sephadex G-50 column(1.5×14 cm), which was equilibrated with 50 mM sodium phosphate, 250 mMNaCl, pH 6.8.

2. Coupling of the Maleoylated BTG with the Peptide

100 μl of the peptide solution (10 mg/ml in 50 mM sodium phosphate, 250mM NaCl, pH 6.8) were mixed with 1 ml of a solution containing themaleoylated carrier proteins (2.5 mg/ml) in 50 mM sodium phosphate, 250ml NaCl, pH 6.8 and incubated for 2 h at 4° C. and further 4 h at RT.Unreacted maleimid groups were blocked by addition of 2-mercaptoethanoleup to a concentration of 10 mM and over night incubation at 4° C. Theresulting conjugate was dialysed against 10 mM sodium phosphate, 150 mMNaCl, pH 7.5 at 4° C. (3 times buffer exchange, MW cut-off 10.000).

Immunization

Five mice were immunized intraperitoneally for 39 days. Forimmunization, a water-in-oil emulsion consisting of equal parts of theantigen solution (consisting of equal parts of the three differentpeptide-BTG-conjugates) and complete or incomplete Freundt's adjuvantswas used.

Fusion

Three of the immunized mice were sacrificed by CO₂ incubation. Spleenswere taken and homogenized under sterile conditions. Spleen cells andmyeloma SP2/0 cells were washed several times in Dulbecco's ModifiedEagle Medium (DMEM, SIGMA) and fused in the ratio of 2,3 spleen cells: 1SP2/0 cell using polyethylenglycole 3350 (1 ml 50% (w/v)). Furtherhandling of the fused hybridomas was performed according to standardmethodologies.

ELISA

An IgG directed ELISA was used to screen the cell culture supernatant.The test was performed in 96-well polystyrol microtiter plates (Greiner,Cat. No. 655061). The plates were coated with the BSA-pGlu-6166 peptide.100 μl undiluted cell culture supernatant was added to each well andincubated for 1 h at RT. Supernatant from SP2/0 cells was used asnegative control. Supernatant from the spleen cells was used as positivecontrol.

Positive wells were detected using goat-anti-mouse IgG, which wasconjugated with alkaline phosphatase. The optical density (OD) wasmeasured in a Dynex Opsys MR Microplate Reader at 405 nm.

Selection of Stable Antibody Producing Hybridoma Cells

Cells from positive wells were transferred to 24-well plates andcultivated for several days. Cells were again transferred and tested forBSA-pGlu-6199 binding and cross-reactivity in the ELISA. Positive wellswere used for cryo-conservation of the hybridoma cell lines.

Cloning via Limited Dilution

Two consecutive cloning steps were performed in order to separateantibody producing cells from non-producing cells and to assure that theselected cells are monoclonal. Both cloning steps were performedaccording to the method of limited dilution.

Cryoconservation

Selected hybridomas were cryo-conserved using DMSO and standard methods.

1.2. ELISA Assays

Capturing of Aβ N3pE-40 was performed using hAp (x-40) ELISA (HS) fromTGC (The GENETICS Company; Switzerland), basically according to themanufacturer's instructions.

Biotinylated detection antibodies for Aβ N3pE (pGlu-6166) weregenerated. Where indicated, IBL HRP-conjugated Aβ N3pE antibody was usedas positive control (available only in combination with the IBL ELISAHuman Amyloid β (N3pE) Assay Kit). Corresponding Aβ N3pE-40 peptides (50μg aliquots in

Hexafluoroisopropanol (HFIP) stored at −80° C.) were synthesized.Shortly before use, HFIP was evaporated and the peptide was diluted with100 mM Tris/HCl pH 10.4 to 1 μg/μl. This stock solution was dilutedfurther with TGC antibody diluent. Subsequent capturing and detectionwas carried out according to manufacturer's instructions.

1.3. PepSpot™ Analysis

Specificity and biological integrity of Aβ N3pE antibodies and cellculture supernatants was determined by using the PepSpot™ technology ofJPT Peptide Technologies GmbH, Volmerstrasse 5 (UTZ), 12489 Berlin,Germany.

Corresponding PepSpot™ membranes were prepared at JPT. The principle ofthis method was introduced and described before by Kramer et al. 1997Cell 91, 799-809.

For analysis, membranes were blocked for two hours with TBST-M (10 mMTris-Hcl, pH 7.5, 150 mM NaCl, 0.005% Tween20+5% skimmed milk) at roomtemperature with gentle shaking. Membranes were incubated over night at4° C. on a rocking platform with the individual cell culturesupernatants diluted in equal volumes of TBST-M. Secondary anti-mouseantibody conjugated with alkaline phosphatase was used for signaldetection, following standard procedures.

1.4. DotBlot Analysis

A simple DotBlot protocol was accomplished to obtain information aboutthe sensitivity of Aβ N3pE antibody and cell culture supernatantstowards the respective native peptide. To that end, Aβ N3pE-40 peptidein descending concentrations (1000 ng-8 ng) was spotted onto smallpieces of nitrocellulose membrane, and subsequent experimental procedurewas performed as for the PepSpot™ membranes.

1.5. SDS PAGE

12% SDS Polyacrylamide gels were cast following standard protocols. 15μl of cell culture supernatants and 10 μg of biotinylated antibody wereseparated on a 12% SDS polyacrylamide gel. Electrophoresis was carriedout for 2 hours at 100 V constant.

1.6. BIACORE Analysis

Aβ N3pE-40 peptide (positive control) and Aβ N3E-40 peptide (negativecontrol) were coupled on a Biacore CM5 Chip. Unmodified chips are usedto determine blank values. Association and dissociation of biotinylatedantibody diluted from 20 μg/ml to 1 μg/ml in TGC diluent was monitoredto allow for subsequent determination of the respective KD value. Inthis way also binding characteristics of the individual cell culturesupernatants were determined.

1.6.1 Affinity of AβN3pE Specific Antibody Clone 6-1-6 and 24-2-3

The purified antibody clone 6-1-6 was diluted in HBS-EP buffer (Biacore)down to 100, 50, 30, 20, 15, 10, 7, 4, 2, 1 nM. The affinity wasdetermined using a Biacore 3000 with CM5-Chip, on which AβpE3-40 wasimmobilized. The system was run with 30 μl/min. Measured bulk effectsand unspecific binding to the chip surface were corrected by subtractionof the signal of flow cell 4, at which AβpE3-40 was immobilized, and theempty flow cell 3. The association (10 min) was obtained by injection of300 μl of each concentration. The dissociation was observed over 10 min.Remaining antibody molecules were removed by injection of 5 μl 0.1 MHCL. For every antibody concentration the association and dissociationwas recorded. The determination of the association and dissociation rateand the dissociation constant was performed by a global simultaneouslyfit of association and dissociation phase for all recorded antibodyconcentrations using the “Bivalent analyte” model.

1.7. Sequencing Antibody Variable Regions Cultivation of HybridomaCells:

Hybridoma cells were grown in D-MEM (+L-Glutamin, +Na-Pyruvat, 4.5 g/lGlucose, Gibco) with the addition of 15% FBS, 1% MEM-NEA (non essentialamino acids, Gibco), 50 μg/ml Gentamycin (Gibco) and 50 μMβ-mercaptoethanol at 37° C. and 5% CO₂. Subcultivation occurred after3-4 days depending on cell density. Cells were seeded in a concentrationof 0.5×10⁶ cells/ml, splitting occurred at a cell density of 2−5×10⁶cells/ml.

cDNA Synthesis and Reverse Transcription:

Total RNA was isolated from 2×10⁶ cells according to the manual of theNucleospinRNA Isolation Kit (Macherey-Nagel). 100 ng RNA were appliedfor cDNA synthesis by using Oligo (dT)₁₅ primer (Promega) andSuperScript III Reverse Transcriptase (Invitrogen).

PCR-Amplification of Heavy and Light Chain Variable Regions:

Heavy chain variable regions were amplified from the template cDNA byusing Phusion™ High-Fidelity DNA Polymerase (NEW ENGLAND BioLabs) withthe primer MHCG1 (in case of clone 5-5-6 and 6-1-6) and MHCG2b (clone17-4-3 and 24-2-3) in combination with primers MHV1-12. Foramplification of light chain variable regions the primer MKC incombination with the primers MKV1-MKV11 were used. Primer sequences areshown in table 1.

Cloning of PCR Products in pJET1.2:

Heavy and light chain variable regions, amplified by PCR, were clonedinto pJET1.2/blunt vector according to the protocol of CloneJET™ PCRCloning Kit (Fermentas). Sequencing occurred with pJET1.2 sequencingprimers.

TABLE 1 Primer sequences for PCR-amplification of heavy and light chainvariable regions Name Sequence SEQ ID NO. MKV1ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG 23 MKV2 ATGGAGWCAGACACACTCCTGYTATGGGTG 24MKV3 ATGAGTGTGCTCACTCAGGTCCTGGSGTTG 25 MKV4ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTT 26 G MKV5ATGGAWWWTCAGGTGCAGATTWTCAGCTTC 27 MKV6 ATGAGGTKCYYTGYTSAGYTYCTGRGG 28MKV7 ATGGGCWTCAAGATGGAGTCACAKWYYCWGG 29 MKV8ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG 30 MKV9 ATGGTRTCCWCASCTCAGTTCCTTG 31MKV10 ATGTATATATGTTTGTTGTCTATTTCT 32 MKV11 ATGGAAGCCCCAGCTCAGCTTCTCTTCC33 MKC ACTGGATGGTGGGAAGATGG 34 MHV1 ATGAAATGCAGCTGGGGCATSTTCTTC 35 MHV2ATGGGATGGAGCTRTATCATSYTCTT 36 MHV3 ATGAAGWTGTGGTTAAACTGGGTTTTT 37 MHV4ATGRACTTTGGGYTCAGCTTGRTTT 38 MHV5 ATGGACTCCAGGCTCAATTTAGTTTTCCTT 39 MHV6ATGGCTTGTCYTRGSGCTRCTCTTCTGC 40 MHV7 ATGGRATGGAGCKGGRTCTTTMTCTT 41 MHV8ATGAGAGTGCTGATTCTTTTGTG 42 MHV9 ATGGMTTGGGTGTGGAMCTTGCTATTCCTG 43 MHV10ATGGGCAGACTTACATTCTCATTCCTG 44 MHV11 ATGGATTTTGGGCTGATTTTTTTTATTG 45MHV12 ATGATGGTGTTAAGTCTTCTGTACCTG 46 MHCG1 CAGTGGATAGACAGATGGGGG 47MHCG2b CAGTGGATAGACTGATGGGGG 48

1.8 Application of Antibody Clone 6-1-6 for N3pE ELISA

A 96-well maxisorb plate (Nunc) was coated with capture antibody byincubation of 100 μl per well of 2 μg/ml anti-Aβ antibody 4G8, dilutedin D-PBS, overnight at 4° C. The plated was sealed. The coating solutionwas removed and the surface of the plate was blocked with 200 μl perwell PIERCE Protein-free ELISA-Blocker (without Tween-20) for 2 hours atroom temperature. Afterwards the plate was washed with 6-times withTBS+0.05% (v/v) Tween-20. Remaining washing solution was removed bytapping of the plate. The AβpE3-40 standard peptide was diluted inPIERCE Protein-free ELISA-Blocker (with Tween-20) down to 200, 100, 50,25, 12.5, 6.25, 3.125 pg/ml. 100 μl of every concentration and 100 μl ofdilution buffer (Blank) were pipetted on the plate. The plate was sealedand incubated at 4° C. for 2 hours. Afterwards the plate was washed6-times with TBS+0.05% (v/v) Tween-20. Remaining washing solution wasremoved by tapping of the plate. 100 μl of the detection antibody-enzymeconjugate solution, which contains 1 μg/ml AβN3pE specific antibodyclone 6-1-6 and 2 μg/ml Streptavidin-HRP conjugate (Sigma) solved inPIERCE Protein-free ELISA-Blocker (with Tween-20), was pipetted in eachwell. The plate was sealed and incubated at 4° C. for 1 hour. Afterwardsthe plate was washed 6-times with TBS+0.05% (v/v) Tween-20. Remainingwashing solution is removed by tapping of the plate. In every well 100μl SureBlue substrate solution (KPL) are pipetted and the plate wasincubated in the dark at room temperature for 30 min. The reaction wasstopped by addition of 100 μl per well of 1 M H₂SO₄. The absorbance wasmeasured with TECAN Sunrise at 450 nm corrected by the absorbance at 540nm.

1.9 Investigation of Cross Reactivity, Analyzed via ELISA andSurface-Plasmon-Resonance (SPR) ELISA:

A 96-well maxisorb plate (Nunc) was coated with capture antibody byincubation of 100 μl per well of 2 μg/ml anti-Aβ antibody 4G8, dilutedin D-PBS, overnight at 4° C. The plated was sealed. The coating solutionwas removed and the surface of the plate was blocked with 200 μl perwell PIERCE Protein-free ELISA-Blocker (without Tween-20) for 2 hours atroom temperature. Afterwards the plate was washed with 6-times withTBS+0.05% (v/v) Tween-20. Remaining washing solution was removed bytapping of the plate. The AβpE3-40 standard peptide and otherAβ-Peptides (2-40, 3-40, 4-40, 1-42, 3-42 and pE11-40) were diluted inPIERCE Protein-free ELISA-Blocker (with Tween-20) down to 800, 400, 200,100, 50, 25, 12.5. 100 μl of every concentration and 100 μl of dilutionbuffer (Blank) were pipetted on the plate. The plate was sealed andincubated at 4° C. for 2 hours. Afterwards the plate was washed 6-timeswith TBS+0.05% (v/v) Tween-20. Remaining washing solution was removed bytapping of the plate. 100 μl of the detection antibody-enzyme conjugatesolution, which contains 1 μg/ml AβN3pE specific antibody clone 6-1-6and 2 μg/ml Streptavidin-HRP conjugate (Sigma) diluted in PIERCEProtein-free ELISA-Blocker (with Tween-20), was pipetted in each well.The plate was sealed and incubated at 4° C. for 1 hour. Afterwards theplate was washed 6-times with TBS+0.05% (v/v) Tween-20. Remainingwashing solution was removed by tapping of the plate. In every well 100μl SureBlue substrate solution (KPL) were pipetted and the plate wasincubated in the dark at room temperature for 30 min. The reaction wasstopped by addition of 100 μl per well of 1 M H₂SO₄. The absorbance wasmeasured with TECAN Sunrise at 450 nm corrected by the absorbance at 540nm.

SPR:

Beside different Aβ species also the cross reactivity to otherpGlu-Peptide, which occur in the human body, was determined. This wasmade by surface plasmon resonance. Following peptides or thereN-terminal region of them were immobilized on the surface of CM5-Chips:

MCP1, MCP2, big gastrin, gonadoliberin, neurotensin, orexin A,fibronectin, collagen 1 and TRH. As positive control also the binding toAβpE3-40 was analyzed. The N3pE antibody clones 6-1-6 and 24-2-3 werediluted in HBS-EP (Biacore) down to 25 μg/ml. The binding was observedusing a Biacore 3000 with several CM5-Chips, on which the respectivepeptides (on flow cell 2, 3 and 4 was immobilized. The system was runwith 20 μl/min. Measured bulk effects and unspecific binding to the chipsurface were corrected by subtraction of the signal of flow cell 2, 3and 4, at which the tested peptides was immobilized, and the empty flowcell 1. The association (9 min) was obtained by injection of 180 μl ofantibody clones 6-1-6 and 24-2-3, respectively. The dissociation wasobserved over 9 min. Remaining antibody molecules were removed byinjection of 5 μl 0.1 M HCL. For every interaction of the antibody withthe different peptides the association and dissociation was recorded.The cross reactivity was determined by evaluation of the associationphase concerning rate and signal at the end. The values for allpGlu-Peptides compared with the signal for AβpE3-40.

1.10 Optimization and Validation of N3pE ELISA for Brain Analysis

Our developed N3pE ELISA should be used for analysis of AβpE3-42concentration in brain of transgenic mice. Generally, hemisphere andbrainstem were separately analyzed concerning AβpE3-42 content. Mousebrain was homogenized in 500 μl 2% SDS solution with protease inhibitorin Precelly (Peqlab) homogenizer using ceramic beads. The suspension waspipetted off from the beads and transferred into centrifuge tube. Beadswere washed again with 250 μl 2% SDS solution with protease inhibitorand solution transferred into the centrifuge tube. The 750 μl SDS brainsuspension was sonificated on crashed ice for 20 sec. The sample wascentrifuged for 1 hour at 4° C. with 75000×g. Afterwards the supernatantwas removed, aliquoted and stored until ELISA analysis at −80° C. Theremaining SDS insoluble pellet was mixed with 150 μl 70% formic acid andsonificated on crashed ice for 20 sec. Immediately after sonificationthe solution was neutralized with 2850 μl 1 M Tris, which was the oldmethod, or 2850 μl EIA buffer (PBS+10 mg/ml BSA+0.05% Tween-20)+860 μl3.5 M Tris for neutralization, representing the new method. The formicacid fraction samples were stored until ELISA at −80° C. The N3pE ELISAwas performed by following protocol:

A 96-well maxisorb plate (Nunc) was coated with capture antibody byincubation of 100 μl per well of 2 μg/ml anti-Aβ antibody 4G8, dilutedin D-PBS, overnight at 4° C. The plated was sealed. The coating solutionwas removed and the surface of the plate was blocked with 200 μl perwell PIERCE Protein-free ELISA-Blocker (without Tween-20) for 2 hours atroom temperature. Afterwards the plate was washed with 6-times withTBS+0.05% (v/v) Tween-20. Remaining washing solution was removed bytapping of the plate. The AβpE3-42 standard peptide was diluted inPIERCE Protein-free ELISA-Blocker (with Tween-20) (old method) or EIAbuffer (new method) down to 1029.2, 514.6, 257.3, 128.65, 64.32, 31.16,16.08 pg/ml. 100 μl of every concentration and 100 μl of dilution buffer(Blank) were pipetted on the plate. The SDS samples were thawed, diluted1:25 and 1:100, respectively, in PIERCE Protein-free ELISA-Blocker (withTween-20) (old method) or EIA buffer (new method) and pipetted on ELISAplate. The formic acid samples (old method: formic acid/Tris; newmethod: formic acid/EIA buffer/Tris) were thawed and undiluted pipettedon ELISA plate. The plate was sealed and incubated at 4° C. for 2 hours.Afterwards the plate was washed 6-times with TBS+0.05% (v/v) Tween-20.Remaining washing solution was removed by tapping of the plate. 100 μlof the detection antibody-enzyme conjugate solution, which contains 1μg/ml AβN3pE specific antibody clone 6-1-6 and 2 μg/ml Streptavidin-HRPconjugate (Sigma) solved in PIERCE Protein-free ELISA-Blocker (withTween-20), was pipetted in each well. The plate was sealed and incubatedat 4° C. for 1 hour. Afterwards the plate was washed 6-times withTBS+0.05% (v/v) Tween-20. Remaining washing solution was removed bytapping of the plate. In every well 100 μl SureBlue substrate solution(KPL) are pipetted and the plate was incubated in the dark at roomtemperature for 30 min. The reaction was stopped by addition of 100 μlper well of 1 M H₂SO₄. The absorbance was measured with TECAN Sunrise at450 nm corrected by the absorbance at 540 nm.

1.11 Application of N3pE Antibody Clones for Immunohistochemistry

Formalin-fixed and paraffin-embedded sections from human brain (cortex)were treated as follows:

1. Deparaffinizing and rehydrating sections (immobilized on slides):

-   -   a. Incubation of slides in Histoclear or xylene for 3 minutes    -   b. Remove cleaning solution    -   c. Incubation of slides again in Histoclear or xylene for 3        minutes    -   d. Incubation of slides in Histoclear or xylene 1:1 with 100%        ethanol for 3 minutes    -   e. Incubation of slides in 100% ethanol for 3 minutes, remove        solution    -   f. Incubation of slides again in 100% ethanol for 3 min    -   g. Incubation of slides in 95% ethanol for 3 minutes    -   h. Incubation of slides in 70% ethanol for 3 minutes    -   i. Incubation of slides in 50% ethanol for 3 minutes    -   j. Incubation of slides in destilled water for 3 minutes        2. Quenching endogenous peroxidase activity:

Incubation of slides with 99 ml methanol+1 ml 30% hydrogen peroxide for10 minutes at room temperature.

3. Washing the slides with water: 2× 5 minutes4. Removing water in individual slides and place slides on slide rack ina humidity chamber to prevent sections from drying. Cover section with88% formic acid at room temperature for 10 minutes under fume hood.Rinse in water several times and allow to shake in a water-filledstaining dish for 10 minutes.5. Blocking in 10% horse serum for 20 minutes at room temperature.6. Shaking off (or aspirate) blocking solution and apply primaryantibody (N3pE antibody clone 6 or 24) for overnight at 4° C.7. Washing slides separately with TBS for 10 minutes to avoid draggingfrom one slide to another.8. Addition of biotinylated secondary antibody (goat anti-mouse fromVector Laboratories): 9 ml TBS, 1 ml goat serum, 45 μl 2^(nd) antibody).Incubate 30 minutes at room temperature.9. Washing slides separately with TBS for 10 minutes to avoid draggingfrom one slide to another.10. Addition of ABC-solution (10 ml TBS, 100 μl horse serum, 90 μlcomponent A, 90 μl component B). Incubate 30 minutes at roomtemperature.11. Washing slides with 50 mM Tris: 2×10 minutes12. Color reaction: Incubation of sections with DAB solution (20 mg DABfrom Sigma in 100 ml 50 mM Tris, filtered, and add 33 μl 33% hydrogenperoxide). Using microscope to observe color reaction. The reactionproduct is brown colored. Stop the reaction by putting slides intostaining dishes containing water.13. Washing slides with water for 10 minutes14. Counterstaining with hematoxylin, washing with water.15. Dehydrating and clearing: Follow step 1 in reverse order (e.g.water, ethanols to 100% histoclean)16. Coverslip with permount (Fisher Scientific). Drying slides on air.Clean slides with razor blade and ethanol.

2. Results 2.1 Production of Antibodies

Six clones were isolated that stably produce antibodies against thepGlu-6166-BSA peptide: clones 1-8-12, 5-5-6, 6-1-6, 12-1-8, 17-4-3 and24-2-3. These clones were subject to further characterization.

2.2. Determination of Required Antibody Concentration:

The intensity of signals in the ELISA assays correlates not only withthe concentration of analyte/Aβ variant but is also strongly dependenton the concentration of deployed antibody. Since Aβ variants are onlypresent in low concentration in serum samples it is necessary todetermine antibody concentrations that are able to detect lowconcentrations of the corresponding Aβ variants. Commercially availableAβ ELISA kits have a specified detection limit towards Aβ in the low pgrange. In standard curves the highest concentration is usually 500pg/ml. General information about deployed antibody concentration istypically lacking in data sheets/instruction manuals, however, due tofurther information as derivable from the general literature 1 μg/ml ofantibody is used as default.

In a first series of experiments it was not possible to detect 500 pg/mlAβ N3pE-40 with the corresponding pGlu-6166 12-1 biotin-conjugatedantibody. In fact, relatively high Aβ N3pE concentrations (10 ng/ml)were required to obtain signals with 1 μg/ml antibody (see FIG. 1A:middle bar). The intensity of this signal was tremendously enhanced byincreasing the antibody concentration to 10 μg/ml (see FIG. 1A:left-hand bar). Until 20 μg/ml antibody the intensity of signal can befurther elevated (see FIG. 1B). Beyond this concentration no furtherincrease in signal intensity can be achieved. Therefore, 20 μg/mlantibody was used to determine the detection limit for the pGlu-6166antibody.

2.3. DotBlot Analysis

The Aβ N3pE-x antibody pGlu-6166 was chosen in the screening processbecause the original cell clone (designated 12-1-8) exhibited strongbinding towards the peptide taken for immunization and very low crossreactivity (see table 2).

TABLE 2 Screening results demonstrating signals in ELISA assays obtainedwith several hybridoma cell clone supernatants. N11E- N11pE- CloneN3pE-BSA isoDAE-BSA N3E-BSA BSA BSA 1-8-12 1.787 0.012 0.142 0.011 0.0055-5-6 1.649 0.015 0.126 0.004 0.006 6-1-6 1.377 0.013 0.125 0.007 0.01412-1-8 2.123 0.005 0.009 0.001 0.005 17-4-3 1.915 0.007 0.320 0.0030.004 24-2-3 1.768 0.014 0.218 0.003 0.002 positive 1.824 1.227 1.5961.243 1.346 control negative 0.045 0.005 0.008 0.001 0.003 control

No screening step, dealing with the full length, native Aβ N3pE-40peptide had been included so far. Therefore the pool of availablepGlu6166 hybridoma cell clones was screened for clones expressingantibodies which might exhibit a higher affinity for the native AβN3pE-40 peptide.

As seen in FIG. 2, cell clones could be identified which indeed exhibithigher sensitivity towards the native full length Aβ N3pE-40 peptide.Whereas the pGlu-6166 antibody clone 12-1 could only detect 1 μgpeptide, clones 6-1-6 and 24-2-3 also gave signals with as little as 8ng peptide. Hence, clones 6-1-6 and 24-2-3 are 125 times more sensitive.With these clones, a detection limit of 8 pg/ml Aβ N3pE-40 peptide in acorresponding ELISA could be attainable.

2.4. PepSpot Analysis

Specificity was checked next by PepSpot analysis to compare biotinylatedAβ N3pE-x antibody pGlu-6166 with hybridoma cell clones. In table 3, allpeptides are listed which correspond to spots on the PepSpot membrane.As seen in FIG. 3 pGlu-6166 clones 6-1-6 and 24-2-3 do not produce morecross signals than pGlu-6166 antibody clone 12-1. All clonesinvestigated recognized primarily spot number 6—the specific Aβ N3pE-xspot (pEFRHD . . . , i.e. SEQ ID No: 12), followed by spots number 5(EFRHD . . . SEQ ID No: 11) and 7 (FRHD . . . SEQ ID No: 13). A faintsignal was also attained with spot number 4 (AEFRHD . . . SEQ ID No:10).

TABLE 3 Sequences of Aβ peptides spotted onto PepSpot ™ Membranes (JPTPeptide Technologies GmbH) and detection by pGlu-6166 hybridoma cellclones Peptide Sequence Aβ 1-40 (SEQ ID NO: 2) No.DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV IBL-AK 5-5-6 6-1-6 17-4-324-2-3 12-4 SEQ ID No. 1 KMDAEFRHDSGYE − − − − +/− −  7 2  DAEFRHDSGYEVH − − − − +/− −  8 3  iDAEFRDHSGYEVH − − − − +/− −  9 4   AEFRHDSGYEVHH − +/− +/− − +/− +/− 10 5     EFRHDSGYEVHHQ − + + + + +11 6    pEFRHDSGYEVHHQ − +++ +++ +++ +++ +++ 12 7     FRHDSGYEVHHQK + + + + + + 13 8           GYEVHHQKLVFFA − − − − − −14 9             EVHHQKLVFFAED − − − − − − 15 10            pEVHHQKLVFFAED − − − − − − 16 11                    VHHQKLVFFAEDV − − − − − − 17 12    DAEFRHiDSGYEVH − −− − − − 18 13          iDSGYEVHHQKLVF − − − − − − 19 14                    LVFFAEDVGSNKG − − − − − − 20 15                            GSNKGAIIGLMVG − − − − − − 21 16                                 AIIGLMVGGVV − − − − − − 22 pE in table 3means pGlu, pyroglutamate. iD in table 3 means isoAsp, isoaspartate.

2.5. SDS-PAGE Analysis

Biological integrity of the Aβ-N3pE antibody and hybridoma cell culturesupernatants was determined roughly by SDS-PAGE analysis (for detailssee Material & Methods supra).

As seen in FIG. 4, all samples loaded onto the gel revealed sharp bandswithout smears, indicating the integrity of the pGlu-6166 12-1 antibodyand the hybridoma cell clone supernatants.

2.6. BIACORE Analysis

With DotBlot analysis significant differences in sensitivity toward theAβ N3pE-40 peptide of hybridoma cell clone supernatants compared tobiotinylated pGlu-6166 antibody were diagnosed. However, with thismethod only an end point result is monitored. Biacore analysis on theother hand allows timewise resolution of the binding course of a givenantibody. In order to check whether the poor binding of the pGlu-616612-1 antibody was a result of a low association to the Aβ N3pE-40peptide, a Biacore analysis was performed as described in Materials andMethods, supra.

Monitoring binding courses of increasing concentrations of pGlu-6166antibody allowed for calculation of a KD value of 30 nM. A comparison ofthe hybridoma cell clone supernatant 12-1 with cell clone supernatant6-1-6 revealed striking differences in binding characteristics. Theassociation of clone 6-1-6 was approximately 5 times higher than thatobserved with clone 12-1. Most markedly, however, is the difference indissociation behavior. Whereas clone 6-1-6 hardly dissociates from theAP N3pE-40 peptide, 12-1 is readily washed off within a few minutes.Hence, the poor binding of clone 12-1 is very likely to be theconsequence of the observed “off-rate”. This assumption is furthersupported by the finding that clone 24-3-2, which gives particularlyadvantageous results in the DotBlot analysis, exhibits a very slowassociation to the Aβ N3pE-40 peptide but—in contrast to clone 12-1—hasno observable “off-rate” (see also FIG. 5).

2.6.1 Affinity of AβN3pE Specific Antibody Clone 6-1-6 and 24-2-3

For N3pE antibody clone 6-1-6 the association rate, dissociation rateand dissociation constant was calculated by a global fit of allsensograms shown in FIG. 6.

The association rate was calculated with 1.67e5 M⁻¹ s⁻¹, thedissociation rate with 2.63e-4 s⁻¹ and the dissociation constant with1.57 nM.

For N3pE antibody clone 24-2-3 the association rate, dissociation rateand dissociation constant was calculated by a global fit of allsensograms shown in FIG. 7.

The association rate was calculated with 3.25e3 M⁻¹ s⁻¹, thedissociation rate with 3.29e-4 s⁻¹ and the dissociation constant with101 nM.

2.7 Sequencing Antibody Variable Regions

The following sequences were identified:

2.7.1 Clone 5-5-6

Variable part light chain, nucleotide sequence (SEQ ID NO: 49)ATGGTGTCCTCAGCTCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGGAAACCAACGGTGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAATGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCAT Variable part light chain, proteinsequence (SEQ ID NO: 50)MVSSAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNWLLQRPGQSPMRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTFGSGTKLEIKRADAAPTVSIFPP Variable part heavy chain,nucleotide sequence (SEQ ID NO. 51)ATGGGATGGAGCGGGGTCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTATACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAGTGGTGTTACTAGGTACAACCAGAAATTCAAGGGCAAGGCCACATTAATTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTATTGTACAAGAGAGGCTAAACGGGAGTGGGACGAGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTA Variable part heavy chain,protein sequence (SEQ ID NO: 52)MGWSGVFLFLLSGTAGVHSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYSGVTRYNQKFKGKATLIVDKSSSTAYMELLSLTSEDSAVYYCTREAKREWDETYWGQGTLVTVSAAKTTPPSV

2.7.2 Clone 6-1-6

Variable part light chain nucleotide sequence (SEQ ID NO: 53)ATGGTGTCCACAGCTCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGGAAACCAACGGTGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGACGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAATGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAG Variable part light chain,protein sequence (SEQ ID NO: 54)MVSTAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNWLLQRPGQSPMRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTFGSGTKLEIKRADAAPTVSIFPPS Variable part heavychain, nucleotide sequence (SEQ ID NO: 55)ATGGGATGGAGCGGGGTCTTTATCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAATGGTGTTACTAGGTACAACCAGAAGTTCAAGGGCAAGGCCACATTAATTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTACAAGAGAGGCTAAACGGGAGTGGGACGAGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTG Variable part heavychain protein sequence (SEQ ID NO: 56)MGWSGVFIFLLSGTAGVHSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGVTRYNQKFKGKATLIVDKSSSTAYMELLSLTSEDSAVYYCTREAKREWDETYWGQGTLVTVSAAKTTPPSVYPL

2.7.3 Clone 17-4-3

Variable part light chain, nucleotide sequence (SEQ ID NO: 57)ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGGTGTTCTGGATTCCTGTTTCCAGCAGTGATGTTGTGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTGATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT Variable part light chain proteinsequence (SEQ ID NO: 58)MKLPVRLLVLVFWIPVSSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPTFGGGTKLEIKRADAAPTVSIFPPSS Variable part heavy chainnucleotide sequence (SEQ ID NO: 59)ATGGACTTTGGGCTCAGCTTACTTATTTTTGTCCTTATTTTAAAAGGTGTCCAGTGTGAGGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTACGGAATGGCGTGGGTTCGACAGGCTCCAGGGAAGGGGCCTGAGTGGGTAGCATTCATTAGTAATTTGGCATATAGTATCTACTATGCAGACACTGTGACGGGCCGATTCACCATCTCTAGAGAGAATGCCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATGTACTACTGTGCAAGGTATGACTACGATAATATCTTGGACTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACACCCCCATCAGTCTAT CCACTG Variable partheavy chain protein sequence (SEQ ID NO: 60)MDFGLSLLIFVLILKGVQCEVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWVRQAPGKGPEWVAFISNLAYSIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARYDYDNILDYVMDYWGQGTSVTVSSAKTTPPSVYPL

2.7.4 Clone 24-2-3

Variable part light chain nucleotide sequence (SEQ ID NO: 61)ATGAAGTTGCCTGTTAGGCTGTTGGTGCTCTGGATTCAGGAAACCAAGGGTGATGTTGTGCTGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATGTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCGTGCAAGGTACACATTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT Variable part light chain proteinsequence (SEQ ID NO: 62)MKLPVRLLVLWIQETKGDVVLTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYVVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTFGSGTKLEIKRADAAPTVSIFPPSS Variable part heavy chainnucleotide sequence (SEQ ID NO: 63)ATGGGATGGAGCGGGGTCTTTCTCTTCCTCCTGTCAGTAACTGAAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTATATATTCAATAACTACTGGATAAACTGGGTGAAGCAGAGGCCTGGTCAGGGTCTTGAGTGGATTGGACAGATTTATCCTGGAGATGGTGATACTAACTACAATGGGAAGTTCAAGGGTAAAGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGAGGGATATATTGTTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTG Variable part heavy chain proteinsequence (SEQ ID NO: 64)MGWSGVFLFLLSVTEGVHSQVQLQQSGAELVRPGSSVKISCKADGYIFNNYWINWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAREGYIVYWGQGTLVTVSAAKTTPPSVYPL2.8 Application of antibody clone 6-1-6 for N3pE ELISA

The final N3pE ELISA protocol was tested concerning limit ofquantification (LOQ) and signal-to-noise ratio (S/N). The standard curveof the ELISA is shown in FIG. 8.

The shape of the standard curve looks very good especially for the lowconcentration range, which shows a nearly linear dependency of theabsorbance. Based on this standard curve the LOQ is determined with3.125 pg/ml with a S/N=1.3.

2.9 Investigation of Cross Reactivity, Analyzed via ELISA and SPR ELISA:

The cross reactivity to other Aβ variants was determined using ourN3pE-ELISA. The raw date are shown in table 4.

TABLE 4 Raw date of N3pE-ELISA with clone 6-1-6: Test of crossreactivity Concen- tration pE3-40 pE3-40 pE11- (pg/ml) (28.04.) (21.04.)2-40 3-40 4-40 1-42 3-42 40 800 1.8280 1.806 0.048 0.090 0.055 0.0530.052 0.047 400 0.8750 0.912 0.045 0.065 0.044 0.048 0.052 0.044 2000.4350 0.484 0.044 0.057 0.046 0.048 0.049 0.048 100 0.2290 0.248 0.0450.052 0.045 0.047 0.050 0.048 50 0.1330 0.143 0.044 0.048 0.044 0.0500.045 0.045 25 0.0820 0.086 0.044 0.046 0.044 0.048 0.047 0.048 12.50.0570 0.063 0.039 0.042 0.040 0.043 0.042 0.046 0 0.0410 0.040 0.0380.065 0.061 0.059 0.063 0.066

Only for AβpE3-40 a dependency of the absorbance from the concentrationwas observed. All tested Aβ variants have shown cross reactivity below1%, except of Aβ3-40. The signal (corrected by the blank) for 800 pg/mlwas about 2.7% of the signal for AβpE3-40. This is a very good value,considering that the N-terminus of both peptides have the same aminoacids, except the first one, this is cyclized in the case of AβpE3-40.Overall, the Aβ N3pE antibody clone 6, which is generally used forELISA, is very high specific for the N-terminus of Aβ-peptides startingwith pGlu at position 3.

SPR:

The cross reactivity of clones 6-1-6 and 24-2-3 to other non-AP pGlupeptides was analyzed by surface plasmon resonance. Instead of AβpE3-40,which shows a typical binding sensogram, all other tested pGlu peptideshave shown nearly no interaction with clones 6-1-6 and 24-2-3,respectively, see also FIG. 9 (data only shown for clone 6-1-6). Thesensograms of the pGlu peptides were compared with the sensogram forAβpE3-40. The estimated cross reactivities were all below 1%. A summaryof all analyzed peptides are shown in table 5.

TABLE 5 Estimated Cross reactivity of clones 6-1-6 and 24-2-3 to otherpGlu-peptides pGlu Peptides % cross reactivity MCP-1 <1 MCP-2 <1 BigGastrin <1 Gonadoliberin <1 Neurotensin <1 Orexin A <1 Fibronectin <1Collagen 1 <1 TRH <1

All experiments have confirmed the fact that N3pE antibody clone 6-1-6and 24-2-3 are specific for the N-terminal epitope of AβpE3-x. Neitherother pGlu N-termini were recognized nor other Aβ peptide variants,which do not bear an N-terminal pE residue.

2.10 Optimization and Validation of N3pE ELISA for Brain Analysis

AβpE3-42 concentration in mouse brainstem was analyzed dependent on theused method. The samples were derived from transgenic mice (tg)overexpressing human AβQ3-42 in the brain, which is cyclized by QC toAβpE3-42. Compared were samples from heterozygous transgenic mice (tghet) and homozygous transgenic mice (tg hom) and from wildtype,non-transgenic mice (wt). The mice used for sample generation wereproduced as described in WO2009034158.

For all further experiments samples and standards were diluted in theEIA buffer. In a next step, the neutralization method for analyzingformic acid fraction samples was optimized, i.e. the neutralization wasN3pE ELISA. The resulting and herein developed N3pE ELISA works well, itdetected significant levels of human AβpE3-42 in brains of the tg hommice, significantly lower levels of human AβpE3-42 in brains of the tghet mice and no human AβpE3-42 in brains of the wt mice (see FIG. 10).The ELISA according to the present invention delivers high signals andconsequently a very acceptable LOQ and is thus suitable for analysis offormic acid samples, in particular of formic acid brain samples.

2.11 Application of N3pE Antibody Clones for Immunohistochemistry

With the N3pE antibodies of the present invention, AβpE3-x was stainedin brain sections of patients in the late stage of sporadic Alzheimer'sdisease (SAD) and familial forms of Alzheimer's disease (FAD), i.e.patients which bear a mutation in the presenilin 1 (PS1) gene. Thestained brain sections are shown in FIG. 11. FIG. 11 shows that the N3pEantibodies of the present invention are suitable forimmunohistochemistry. The antibodies specifically detect pGlu-Aβ inbrain of SAD and FAD patients. The N3pE antibodies show no backgroundsignals on the images, which proves the specific binding shown by ELISAand SPR analysis.

3. Deposits

Monoclonal antibodies specifically recognizing Aβ N3pE-x, weregenerated. Currently all corresponding monoclonal antibodies expressinghybridoma cell lines 5-5-6, 6-1-6, 17-4-3, and 24-2-3 have beendeposited in accordance with the Budapest Treaty and are available atthe Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ) inBraunschweig, DE, with a deposit date of Jun. 17, 2008, and with therespective deposit numbers

(clone 5-5-6) DSM ACC2923 (clone 6-1-6) DSM ACC2924 (clone 17-4-3) DSMACC2925 (clone 24-2-3) DSM ACC2926.

Specificity of those antibodies for their respective target sequencescould be confirmed. For Aβ N3pE-x, high affinity antibody clones couldbe identified that should give strong signals in an ELISA set up with anexpected detection limit in the low pg range.

4. Summary

One objective of the present invention was the establishment of a highlysensitive and robust detection technique that allows quantitativedetermination of Aβ variants in biological samples.

Preferably, an ELISA based technique can be pursued. The task wasstarted with Aβ N3pE ELISA, because for this Aβ variant an appropriateELISA system was already commercially available (IBL). This system wasused as reference and internal quality control.

Applicability of the pGlu-6166 antibody in the chosen ELISA assay set upwas investigated. To obtain clearly measurable signals, high antibodyconcentrations needed to be deployed (20 μg/ml). High affinity Aβ N3pE-xantibody clones could be identified. A detection limit in the low pgrange (3-8 pg/ml) can be achieved with these clones.

1. An isolated antibody capable of binding to an Aβ peptide or variantthereof, optionally with high affinity.
 2. The antibody according toclaim 1, wherein the antibody binds an Aβ peptide with a dissociationconstant (K_(D)) value of at least 10⁻⁷ M.
 3. The antibody according toclaim 1, wherein said antibody is a monoclonal antibody.
 4. The antibodyaccording to claim 1, wherein: the isolated antibody comprises a lightchain; the light chain comprises a variable part; and the variable partof the light chain of said antibody comprises a polypeptide encoded by anucleotide sequence selected from SEQ ID NOs: 49, 53, 57 and 61, orhaving an amino acid sequence selected from SEQ ID NOs: 50, 54, 58, and62.
 5. The antibody according to claim 1, wherein: the isolated antibodycomprises a heavy chain; the heavy chain comprises a variable part; andthe variable part of the heavy chain of said antibody comprises apolypeptide encoded by a nucleotide sequence selected from SEQ ID NOs:51, 55, 59 and 63, or having an amino acid sequence selected from SEQ IDNOs: 52, 56, 60 and
 64. 6. The antibody according to claim 1, wherein:the isolated antibody comprises a light chain; the light chain comprisesa variable part; the variable part of the light chain of said antibodycomprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO:49 or having an amino acid sequence of SEQ ID NO: 50; the isolatedantibody comprises a heavy chain; the heavy chain comprises a variablepart; and the variable part of the heavy chain of said antibodycomprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO:51, or having an amino acid sequence of SEQ ID NO:
 52. 7. The antibodyaccording to claim 1, wherein: the isolated antibody comprises a lightchain; the light chain comprises a variable part; the variable part ofthe light chain of said antibody comprises a polypeptide encoded by anucleotide sequence of SEQ ID NO: 53 or having an amino acid sequence ofSEQ ID NO: 54; the isolated antibody comprises a heavy chain; the heavychain comprises a variable part; and the variable part of the heavychain of said antibody comprises a polypeptide encoded by a nucleotidesequence of SEQ ID NO: 55, or having an amino acid sequence of SEQ IDNO:
 56. 8. The antibody according to claim 1, wherein: the isolatedantibody comprises a light chain; the light chain comprises a variablepart; the variable part of the light chain of said antibody comprises apolypeptide encoded by a nucleotide sequence of SEQ ID NO: 57 or havingan amino acid sequence of SEQ ID NO: 58; the isolated antibody comprisesa heavy chain; the heavy chain comprises a variable part; and thevariable part of the heavy chain of said antibody comprises apolypeptide encoded by a nucleotide sequence of SEQ ID NO: 59, or havingan amino acid sequence of SEQ ID NO:
 60. 9. The antibody according toclaim 1, wherein: the isolated antibody comprises a light chain; thelight chain comprises a variable part; the variable part of the lightchain of said antibody comprises a polypeptide encoded by a nucleotidesequence of SEQ ID NO: 61 or having an amino acid sequence of SEQ ID NO:62; the isolated antibody comprises a heavy chain; the heavy chaincomprises a variable part; and the variable part of the heavy chain ofsaid antibody comprises a polypeptide encoded by a nucleotide sequenceof SEQ ID NO: 63, or having an amino acid sequence of SEQ ID NO:
 64. 10.The antibody according to claim 1, wherein said antibody is selectedfrom the group consisting of: Aβ 5-5-6 (Deposit No. DSM ACC 2923); Aβ6-1-6 (Deposit No. DSM ACC 2924); Aβ 17-4-3 (Deposit No. DSM ACC 2925);and Aβ 24-2-3 (Deposit No. DSM ACC 2926); or functional variantsthereof.
 11. The antibody according to claim 1, wherein said antibody isAβ 6-1-6 (Deposit No. DSM ACC 2924).
 12. The antibody according to claim1, wherein said antibody is Aβ 24-2-3 (Deposit No. DSM ACC 2926). 13.The antibody according to claim 1, wherein said antibody is a humanizedor chimeric antibody, or an antibody fragment which retains the highaffinity.
 14. The antibody according to claim 1, wherein the antibody isa human antibody.
 15. The antibody according to claim 1, wherein theantibody is a diabody or a single chain antibody which retains the highaffinity.
 16. The antibody according to claim 1, wherein the antibody iscapable of binding an epitope of an antigen selected from the groupconsisting of: pGlu-Aβ₃₋₃₈; pGlu-Aβ₃₋₄₀; pGlu-Aβ₃₋₄₂; and pGlu-Aβ₃—,variants; wherein x is an integer between 10 and 42; preferably 18 and42, more preferably 30 and
 42. 17. The antibody according to claim 1,wherein the antibody comprises a complementarity determining regioncapable of binding to an antigen selected from the group consisting of:pGlu-Aβ₃₋₃₈; pGlu-Aβ₃₋₄₀; pGlu-Aβ₃₋₄₂; and pGlu-Aβ₃-x variants; whereinx is an integer between 10 and 42; preferably 18 and 42, more preferably30 and
 42. 18. The antibody according to claim 1, wherein the antibodyis labeled.
 19. The antibody according to claim 1, wherein the antibodyis immobilised on a solid phase.
 20. The antibody according to claim 1,wherein the antibody obtainable from any one of hybridoma cell lines DSMACC 2923, DSM ACC 2924, DSM ACC 2925, or DSM ACC
 2926. 21. An isolatednucleic acid comprising a nucleotide sequence selected from the groupconsisting of SEQ ID No: 23, SEQ ID No: 24; SEQ ID No: 25; SEQ ID No:26; SEQ ID No: 27; SEQ ID No: 28; SEQ ID No: 29; SEQ ID No: 30; SEQ IDNo: 31; SEQ ID No: 32; SEQ ID No: 33; SEQ ID No: 34; SEQ ID No: 35; SEQID No: 36; SEQ ID No: 37; SEQ ID No: 38; SEQ ID No: 39; SEQ ID No: 40;SEQ ID No: 41; SEQ ID No: 42; SEQ ID No: 43; SEQ ID No: 44; SEQ ID No:45; SEQ ID No: 46; SEQ ID No: 47; and SEQ ID No:
 48. 22. A compositioncomprising a hybridoma cell line selected from the group consisting ofDSM ACC 2923; DSM ACC 2924; DSM ACC 2925; and DSM ACC
 2926. 23. Acomposition comprising the antibody of claim 1 and, optionally, apharmaceutically acceptable carrier.
 24. A method for treatment,prevention or delay of amyloidosis comprising administering atherapeutically effective amount of the composition of claim 23 to asubject in need thereof.
 25. The method of claim 24, wherein saidamyloidosis is a neurodegenerative disease selected from the groupconsisting of mild cognitive impairment, Alzheimer's disease andneurodegeneration in Down Syndrome.
 26. The method of claim 24, whereinsaid amyloidosis is sporadic Alzheimer's disease or a FamilialAlzheimer's dementia.
 27. The method of claim 24, wherein said FamilialAlzheimer's dementia is Familial British Dementia or Familial DanishDementia.
 28. A method for detecting an Aβ peptide or variants thereofcomprising: contacting an antibody of claim 1 and a sample potentiallycomprising an Aβ peptide or variants thereof; and detecting binding ofthe antibody and the Aβ peptide or variant thereof.
 29. The methodaccording to claim 28, wherein said Aβ peptide variants are selectedfrom the group consisting of: pGlu-Aβ₃₋₃₈; pGlu-Aβ₃₋₄₀; pGlu-Aβ₃₋₄₂; andpGlu-Aβ_(3-x) variants; wherein x is an integer between 10 and 42; 18and 42; or and
 42. 30. A method for in vitro diagnosis of anamyloid-associated disease or condition, optionally Alzheimer's disease,comprising: contacting an antibody according to claim 1 and a samplefrom a subject suspected to be afflicted with said disease or condition,and detecting binding of the antibody to a pGlu-amyloid protein,optionally pGlu-Aβ peptide, from the sample.
 31. A diagnostic kitcomprising the antibody according to claim 1 and instructions for use,and, optionally, at least one additional biologically active substance.32. The diagnostic kit according to claim 31, wherein said at least oneadditional biologically active substance comprises an inhibitor ofglutaminy cyclase.